Bispecific anti-CD37 antibodies, monoclonal anti-CD37 antibodies and methods of use thereof

ABSTRACT

CD37-specific bispecific antibody molecules binding to different epitopes of the human CD37 antigen which bispecific antibody molecules have enhanced Fc-Fc interactions upon binding to CD37 on the cell surface. The invention also relates to the monoclonal parental antibodies from which the first or the second binding region of the bispecific antibody molecules is obtained. The invention also relates to pharmaceutical compositions containing these molecules and the treatment of cancer and other diseases using these compositions.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.16/872,140, filed May 11, 2020, which is a continuation of U.S. patentapplication Ser. No. 16/498,104, filed Sep. 26, 2019, now abandoned,which is a 35 U.S.C. 371 national stage filing of InternationalApplication No. PCT/EP2018/058479, filed Apr. 3, 2018, which claimspriority to International Application No. PCT/EP2018/057836, filed Mar.27, 2018, and U.S. Provisional Application No. 62/479,712, filed Mar.31, 2017.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference it is entirety. Said ASCII copy, created on Jul. 22, 2021, isnamed GMI_162USCNDV2_Sequence_Listing.txt and is 109,337 bytes in size.

FIELD OF THE INVENTION

The present invention relates to bispecific antibodies that specificallybind the human CD37 antigen. The invention relates in particular toCD37-specific bispecific antibody molecules binding to differentepitopes of the human CD37 antigen where the bispecific antibodymolecules have enhanced Fc-Fc interactions upon binding to CD37 on thecell surface and thus have enhanced effector functions. The inventionalso relates to new monoclonal parental antibodies from which the firstor the second antigen binding region of the bispecific antibodymolecules is obtained. The invention also relates to pharmaceuticalcompositions containing these molecules and the treatment of cancer andother diseases using these compositions.

BACKGROUND OF THE INVENTION

Leukocyte antigen CD37 (“CD37”), also known as GP52-40, tetraspanin-26,or TSPAN26, is a transmembrane protein of the tetraspanin superfamily(Maecker et al., FASEB J. 1997; 11:428-442). In normal physiology, CD37is expressed on B cells during the pre-B to peripheral mature B-cellstages but is reportedly absent on plasma cells (Link et al., J Pathol.1987; 152:12-21). The CD37 antigen is only weakly expressed on T-cellsand myeloid cells such as monocytes, macrophages, dendritic cells andgranulocytes (Schwartz-Albiez et al., J. Immunol 1988; 140(3):905-914).CD37 is broadly expressed on malignant cells in a variety of B-cellleukemias and lymphomas, including non-Hodgkin's lymphoma (NHL) andchronic lymphoid leukemia (CLL) (Moore et al. J Immunol. 1986;137(9):3013).

Several antibody-based CD37-targeting agents are being evaluated aspotential therapeutics for B-cell malignancies and other malignancies.These include, for example, radio-immuno-conjugates such as Betalutin®,antibody-drug conjugates such as IMGN529 and AGS-67E, and reformatted orFc-engineered antibodies such as otlertuzumab and BI 836826 (Robak andRobak, Expert Opin Biol Ther 2014; 14(5):651-61). Anti-CD37 antibodieshave been proposed for use as therapeutic agents in the formatsdescribed above and other formats (see, e.g., WO 2012/135740, WO2012/007576, WO 2011/112978, WO 2009/126944, WO 2011/112978 and EP2241577).

Betalutin is a mouse anti-CD37 antibody, lilotomab (formerlyHH1/tetulomab), conjugated to 177-lutetium. Betalutin internalizesrapidly, inhibits B cell growth in vitro and prolongs survival in ani.v. Daudi-SCID model (Dahle et al 2013, Anticancer Res 33: 85-96).

IMGN529 is an ADC consisting of the K7153A antibody conjugated to themaytansinoid DM1 via an SMCC linker. The K7153 antibody is reported toinduce apoptosis on CD37 expressing Ramos cells in the absence ofcross-linking. It also induced CDC and ADCC in Burkitt's lymphoma celllines, though the ability to induce CDC was much less compared torituximab (Deckert et al, Blood 2013; 122(20):3500-10). TheseFc-mediated effector functions of K7153A are retained in the DM-1conjugated antibody.

Agensys is developing AGS-67E, a human anti-CD37 IgG2 mAb conjugated tomonomethyl auristatin E. AGS67E induces potent cytotoxicity andapoptosis (Pereira et al, Mol Cancer Ther 2015; 14(7): 1650-1660).

Otlertuzumab (originally known as TRU-016) is a SMIP (small modularimmuno pharmaceutical; SMIPS are disulfide-linked dimers of single-chainproteins comprised of one antigen binding VH/VL, a connecting hingeregion, and an Fc (fragment, crystallizable) region (CH2-CH3)). Itsmechanisms of action are induction of apoptosis and ADCC, but not CDC(Zhao et al 2007, Blood 110 (7), 2569-2577).

mAb37.1/BI 836826 is a chimeric antibody that is engineered forhigh-affinity binding to FcγRIIIa (CD16a) (Heider et al 2011, Blood 118:4159-4168). It has pro-apoptotic activity independent of IgG Fccrosslinking, although the pro-apoptotic activity is increased bycross-linking. It shows potent ADCC of CD37+B cell lines and primary CLLcells.

Despite these and other advances in the art, however, there is still aneed for improved anti-CD37 antibodies for the treatment of cancer andother diseases.

Accordingly, it is an object of the present invention to provideanti-CD37 antibodies which may be useful in the treatment of cancerand/or other diseases. It is an object of the present invention toprovide anti-CD37 antibodies which are improved with respect to CDC ofhuman cells by human complement compared to the prior art antibodies. Itis a further object to provide a bispecific antibody having binding armsobtained from two parental antibodies which bind to different epitopeson CD37 and which bispecific antibody has increased CDC and/or ADCCcompared to a combination of the two parental monoclonal antibodiesbinding said different epitopes, and/or to either parental monoclonalantibody by itself. It is a further object to provide new monoclonalantibodies binding different epitopes on CD37 in particular it is anobject to provide anti-CD37 antibodies binding new epitopes of CD37. Itis a further object of the present invention to provide new monoclonalantibodies binding different epitopes on CD37 which monoclonalantibodies may serve as parental antibodies for the bispecificantibodies of the invention. It is a further object to providebispecific antibodies which bind to two different epitopes on CD37 andwhich bispecific antibodies have enhanced Fc-Fc interaction upon bindingto CD37 on the plasma membrane compared to a bispecific antibody of thesame isotype and having identical binding arms as the bispecificantibody of the invention.

SUMMARY OF THE INVENTION

The inventors of the present invention surprisingly found that abispecific antibody having binding specificity against two differentepitopes on CD37 and having a mutation increasing the Fc-Fc interactionupon binding to CD37 on the plasma membrane was more potent in inducingCDC than a combination of two anti-CD37 antibodies each having a bindingspecificity towards one of the two different epitopes on CD37 and havingthe same mutation enhancing the Fc-Fc interaction, or either antibodyhaving the same mutation enhancing the Fc-Fc interaction by itself. Inaddition, a bispecific antibody having binding specificity against twodifferent epitopes on CD37 and having a mutation increasing the Fc-Fcinteractions was more potent in inducing ADCC than a combination of twoanti-CD37 antibodies each having a binding specificity towards one ofthe two different epitopes on CD37 and having the same mutationenhancing the Fc-Fc interaction.

Accordingly, the invention relates to novel bispecific antibodiesbinding to human CD37 which have advantageous properties in terms oftheir antigen-binding characteristics, their ability to induce CDC andADCC, their Fc-Fc interaction upon binding to membrane-bound targets,their cytotoxic effect on CD37-expressing cells and other properties, asdescribed herein.

Accordingly, in a first aspect the present invention relates to abispecific antibody comprising a first and second antigen binding regionbinding to human CD37 having the sequence of SEQ ID NO: 62, and a firstand second Fc region of a human immunoglobulin, wherein the first andsecond antigen binding regions bind different epitopes on CD37 andwherein the first and second Fc regions comprises one or more amino acidmutations which mutation(s) enhances the Fc-Fc interaction between thebispecific antibodies upon binding to membrane-bound CD37 compared tothe Fc-Fc interaction between bispecific antibodies not having saidmutation(s).

Thus, in one aspect a bispecific antibody comprising a first and secondantigen binding region binding to human CD37 having the sequence of SEQID NO: 62, and a first and second Fc region of a human immunoglobulin,wherein the first and second antigen binding regions bind differentepitopes on CD37 and wherein the first and second Fc regions compriseone or more amino acid mutations which mutation(s) enhances the Fc-Fcinteraction between bispecific antibodies upon binding to membrane-boundtarget compared to the Fc-Fc interaction between bispecific antibodiesnot having said mutation(s).

In a second aspect the invention relates to an anti-CD37 antibodybinding to the same epitope on human CD37 as an anti-CD37 antibody whichantibody comprises:

-   -   (i) a VH region comprising a CDR1 sequence set forth in SEQ ID        NO: 16, a CDR2 sequence set forth in SEQ ID NO: 17 and a CDR3        sequence set forth in SEQ ID NO: 18, and a VL region comprising        a CDR1 sequence set forth in SEQ ID NO: 20, and CDR2 sequence:        KAS, and CDR3 sequence set forth in SEQ ID NO: 21[010]; or    -   (ii) a VH region comprising a CDR1 sequence set forth in SEQ ID        NO: 9, a CDR2 sequence set forth in SEQ ID NO:10 and a CDR3        sequence set forth in SEQ ID NO: 11, and a VL region comprising        a CDR1 sequence set forth in SEQ ID NO: 113, and CDR2 sequence:        AAS, and CDR3 sequence set forth in SEQ ID NO: 14[005].

In a third aspect the invention relates to an anti-CD37 antibody whichbinds to human CD37 which antibody comprises:

-   -   (i) a VH region comprising a CDR1 sequence set forth in SEQ ID        NO: 23, a CDR2 sequence set forth in SEQ ID NO: 24 and a CDR3        sequence set forth in SEQ ID NO: 25, and a VL region comprising        a CDR1 sequence set forth in SEQ ID NO: 27, and CDR2 sequence:        YAS, and CDR3 sequence set forth in SEQ ID NO: 28; [016] or    -   (ii) a VH region comprising a CDR1 sequence set forth in SEQ ID        NO: 2, a CDR2 sequence set forth in SEQ ID NO: 3 and a CDR3        sequence set forth in SEQ ID NO: 4, and a VL region comprising a        CDR1 sequence set forth in SEQ ID NO: 6, and CDR2 sequence: EAS,        and CDR3 sequence set forth in SEQ ID NO: 7. [004]

In a fourth aspect the invention relates to a pharmaceutical compositioncomprising the bispecific antibody or the antibody of the invention anda pharmaceutically acceptable carrier.

In a fifth aspect the invention relates to the bispecific antibody orthe antibody or the composition of the invention for use as amedicament. In specific aspects they are for use in the treatment ofcancer or an autoimmune disease or inflammatory disorders and inparticular for use in the treatment of B-cell malignancies.

In other aspects the invention relates to methods of treatment, tocombination treatments, to nucleic acid sequences encoding theantibodies of the invention, to vectors and host cells expressing suchand to methods of detecting presence or a CD37 antigen or cellsexpressing CD37 antigens in a sample or in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B: CDC mediated by G28.1 variants on primary CLL tumorcells. The capacity to induce CDC on primary CLL tumor cells of (FIG.1A) IgG1-G28.1-K409R-delK, IgG1-G28.1-E345R or IgG1-b12-E345R (cells:Patient derived, Newly Diagnosed/Untreated (PB=peripheral bloodderived)) and (FIG. 1B) IgG1-G28.1, IgG1-G28.1-E430G or IgG1-b12 (cells:Patient derived, Newly Diagnosed/Untreated (BM=bone marrow derived)) wasdetermined in vitro. Data shown are % lysis determined by measurement ofthe percentage of dead cells (corresponding to PI-positive cells) byflow cytometry.

FIG. 2: Quantitative determination of CD37, CD46, CD55 and CD59expression levels on CLL tumor cells. Expression levels of CD37, CD46,CD55 and CD59 on CLL cells from one patient (Patient VM-PB0005 NewlyDiagnosed/Untreated) were determined by flow cytometry. Antigen quantityis shown as molecules/cell. mIgG1 is Mouse IgG1,κ Isotype Control.

FIG. 3: Binding of humanized CD37 antibodies and variants thereof toDaudi cells. Binding of IgG1-004-H5L2, IgG1-004-H5L2-E430G,IgG1-005-H1L2, IgG1-005-H1L2-E430G, IgG1-010-H5L2, IgG1-010-H5L2-E430G,IgG1-016-H5L2 and IgG1-016-H5L2-E430G to Daudi cells was determined byflow cytometry. Data shown are mean fluorescence intensity (MFI) values,for one representative experiment.

FIG. 4: Binding of G28.1 and 37.3 and variants thereof to Daudi cells.Binding of IgG1-G28.1, IgG1-G28.1-E430G, IgG1-37.3 and IgG1-37.3-E430Gto Daudi cells was determined by flow cytometry. Data shown are meanfluorescence intensity (MFI) values, for one representative experiment.

FIG. 5: Binding of variants of humanized CD37 antibody IgG1-016-H5L2 toDaudi cells. Binding of IgG1-016-H5L2, IgG1-016-H5L2-E430G,IgG1-016-H5L2-F405L-E430G and IgG1-016-H5L2-LC90S-F405L-E430G to Daudicells was determined by flow cytometry. Data shown are mean fluorescenceintensity (MFI) values, for one representative experiment.

FIG. 6: Binding of CD37 antibody variants to CHO cells expressingcynomolgus CD37. Binding of IgG1-004-H5L2-E430G, IgG1-005-H1L2-E430G,IgG1-010-H5L2-E430G, IgG1-016-H5L2-E430G, IgG1-G28.1 andIgG1-G28.1-E430G was determined by flow cytometry. Data shown are meanfluorescence intensity (MFI) values, for one representative experiment.

FIGS. 7A-7G: Determination of binding competition between CD37antibodies, and CDC mediated by humanized CD37 antibodies, variantsthereof and combinations of CD37 antibodies on Raji cells. (FIG. 7A)Binding competition between IgG1-37.3-E430G, IgG1-G28.1-E430G,IgG1-004-H5L2-E430G, IgG1-005-H1L2-E430G, IgG1-010-H5L2-E430G andIgG1-016-H5L2-E434G was determined by flow cytometry. Raji cells wereincubated with unlabeled antibodies for primary binding and subsequentlywith Alexa Fluor 488 labeled probing antibodies. Loss of binding of theA488-labeled probing antibodies after pre-incubation with an unlabeledantibody, compared to binding of the A488-labeled antibody alone,indicates binding competition between the A488-labeled and the unlabeledantibody. Data shown are duplicate values of Molecules of EquivalentSoluble Fluorochrome (MESF), for one representative experiment. (FIGS.7B-7G) The capacity to induce CDC on Raji cells of IgG1-004-H5L2, ofIgG1-005-H1L2, IgG1-010-H5L2, IgG1-016-H5L2 and IgG1-37.3, with orwithout E430G mutation, and combinations of these was determined invitro. Data shown are % lysis determined by measurement of thepercentage of dead cells (corresponding to PI-positive cells) by flowcytometry.

FIG. 8: Schematic overview of binding competition between CD37antibodies. Binding competition between IgG1-37.3-E430G,IgG1-G28.1-E430G, IgG1-004-H5L2-E430G, IgG1-005-H1L2-E430G,IgG1-010-H5L2-E430G and IgG1-016-H5L2-E4340G to Raji cells wasdetermined by flow cytometry, using unlabeled antibodies for primarybinding and Alexa Fluor 488 labeled probing antibodies for detectingsubsequent binding of a competing antibody. Color indication: black;simultaneous binding, white; competition for binding, grey; cognateantibody.

FIG. 9: CDC mediated by humanized CD37 antibodies and variants thereofon Daudi cells. The capacity to induce CDC on Daudi cells ofIgG1-004-H5L2, IgG1-004-H5L2-E430G, IgG1-005-H1L2, IgG1-005-H1L2-E430G,IgG1-010-H5L2, IgG1-010-H5L2-E430G, IgG1-016-H5L2 andIgG1-016-H5L2-E430G was determined in vitro. Data shown are % lysisdetermined by measurement of the percentage of dead cells (correspondingto PI-positive cells) by flow cytometry.

FIGS. 10A-10C: CDC mediated by G28.1 and 37.3 and variants thereof, andCDC in Daudi cells mediated by humanized CD37 antibodies with differentFc-Fc interaction enhancing mutations on Daudi cells. (FIG. 10A) Thecapacity to induce CDC on Daudi cells of IgG1-G28.1, IgG1-G28.1-E430G,IgG1-37.3 and IgG1-37.3-E430G was determined in vitro. Data shown are %lysis determined by measurement of the percentage of dead cells(corresponding to PI-positive cells) by flow cytometry. (FIGS. 10B and10C) The capacity to induce CDC on Daudi cells of (FIG. 10A)IgG1-010-H5L2-K409R-E430G, IgG1-010-H5L2-E345R-K409R,IgG1-010-H5L2-E345K-K409R, IgG1-010-H5L2-K409R-E430S, IgG1-010-H5L2-RRGYand (FIG. 10B) IgG1-016-H5L2-LC90S-F405L-E430G,IgG1-016-H5L2-E345K-F405L, IgG1-016-H5L2-F405L-E430S andIgG1-016-H5L2-E345R-F405L was determined in vitro. Data shown are %lysis (maximum killing, at an antibody concentration of 10 μg/mL)determined by measurement of the percentage of dead cells (correspondingto PI-positive cells) by flow cytometry, for one representativeexperiment. Error bars indicate the variation within the experiment(performed in duplicate).

FIG. 11: CDC mediated by variants of humanized antibody IgG1-016-H5L2 onDaudi cells. The capacity to induce CDC on Daudi cells of IgG1-016-H5L2,IgG1-016-H5L2-E430G, IgG1-016-H5L2-F405L-E430G andIgG1-016-H5L2-LC90S-F405L-E430G was determined in vitro. Data shown are% lysis determined by measurement of the percentage of dead cells(corresponding to PI-positive cells) by flow cytometry.

FIGS. 12A-12E: CDC mediated by bispecific CD37 antibodies with an Fc-Fcinteraction enhancing mutation, (combinations of) CD37 antibodies withan Fc-Fc interaction enhancing mutation, and monovalent CD37-bindingantibodies with an Fc-Fc interaction enhancing mutation on Daudi cells;and CDC activity of CD37 antibody variants with an Fc-Fc interactionenhancing mutation, and combinations thereof, on OCI-Ly-7 cells. (FIG.12A) The capacity to induce CDC on Daudi cells ofbsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G,IgG1-005-H1L2-E430G, IgG1-016-H5L2-E430G, a combination ofIgG1-005-H1L2-K409R-E430G plus IgG1-016-H5L2-F405L-E430G,bsIgG1-b12-F405L-E430Gx005-H1L2-K409R-E430G andbsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G, and (FIG. 12B)bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G,IgG1-010-H5L2-E430G, IgG1-016-H5L2-E430G, a combination ofIgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G,bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G andbsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G was determined in vitro.Data shown are % lysis determined by measurement of the percentage ofdead cells (corresponding to PI-positive cells) by flow cytometry. (FIG.12C) The capacity to induce OCI-Ly-7 cells of the CD37 bispecificantibody bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G, theCD37 monospecific bivalent (monoclonal) antibodies IgG1-010-H5L2-E430G,IgG1-016-H5L2-E430G, a combination of IgG1-010-H5L2-E430G plusIgG1-016-H5L2-E430G, the monovalent CD37 antibodiesbsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G,bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G and a combination ofbsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G plusbsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G was determined in vitro.Data shown are % lysis determined by measurement of the percentage ofdead cells (corresponding to PI-positive cells) by flow cytometry. (FIG.12D) EC50 values of CDC induction bybsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G plusbsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G and IgG1-010-H5L2-E430G plusIgG1-016-H5L2-E430G, as determined in 2 independent experiments. (FIG.12E) EC50 values of CDC induction bybsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G andIgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G, as determined in 3independent experiments.

FIGS. 13A and 13B: CDC mediated by bispecific CD37 antibodies and bybispecific CD37 antibodies with an Fc-Fc interaction enhancing mutationon Daudi cells. The capacity to induce CDC on Daudi cells of (FIG. 13A)bsIgG1-016-H5L2-F405Lx005-H1L2-K409R andbsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G, and (FIG. 13B)bsIgG1-016-H5L2-F405Lx010-H5L2-K409R andbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G was determined invitro. Data shown are % lysis determined by measurement of thepercentage of dead cells (corresponding to PI-positive cells) by flowcytometry.

FIGS. 14A and 14B: CDC mediated by bispecific CD37 antibodies with anFc-Fc interaction enhancing mutation, (combinations of) CD37 antibodieswith an Fc-Fc interaction enhancing mutation, and monovalent bindingCD37 antibodies with an Fc-Fc interaction enhancing mutation on primaryCLL tumor cells. The capacity to induce CDC on primary CLL tumor cells(Patient: VM-BM0091 Newly Diagnosed/Untreated (BM=bone marrow derived))of (FIG. 14A) bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G,IgG1-005-H1L2-K409R-E430G, IgG1-016-H5L2-F405L-E430G, a combination ofIgG1-005-H1L2-K409R-E430G plus IgG1-016-H5L2-F405L-E430G,bsIgG1-b12-F405L-E430Gx005-H1L2-K409R-E430G andbsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G, and (FIG. 14B)bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G,IgG1-010-H5L2-E430G, IgG1-016-H5L2-E430G, a combination ofIgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G,bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G andbsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G was determined in vitro.Data shown are % lysis determined by measurement of the percentage ofdead cells (corresponding to PI-positive cells) by flow cytometry.

FIG. 15: CDC mediated by a bispecific CD37 antibody with an Fc-Fcinteraction enhancing mutation on B cell lymphoma cell lines. Thecapacity of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G, at aconcentration of 10 μg/mL, to induce CDC on a range of B cell lymphomacell lines was determined in vitro. Expression levels of CD37 weredetermined by quantitative flow cytometry, and are shown asmolecules/cell, average±SD of 2 experiments. White bars indicatesusceptible to CDC (>10% lysis, average of 2 experiments) mediated bybsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G, black barsindicate unsusceptible to CDC (<10% lysis, average of 2 experiments)mediated by bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G.

FIGS. 16A-16C: ADCC mediated by bispecific CD37 antibodies with an Fc-Fcinteraction enhancing mutation, (combinations of) CD37 antibodies withan Fc-Fc interaction enhancing mutation, and monovalent binding CD37antibodies with an Fc-Fc interaction enhancing mutation on Daudi andRaji cells. The capacity to induce ADCC of (FIG. 16A)bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G,IgG1-005-H1L2-K409R-E430G, IgG1-016-H5L2-F405L-E430G and a combinationof IgG1-005-H1L2-K409R-E430G plus IgG1-016-H5L2-F405L-E430G on Daudicells, and (FIG. 16B)bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G,IgG1-010-H5L2-E430G, IgG1-016-H5L2-E430G and a combination ofIgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G on Daudi cells, and (FIG.16C) bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G,IgG1-010-H5L2-E430G, IgG1-016-H5L2-E430G, a combination ofIgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G,bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G andbsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G on Raji cells was determinedin vitro using a chromium release assay. Data shown are % specificlysis; error bars indicate variation within the assay, with 5 replicates(FIG. 16A, FIG. 16B) or 6 replicates (FIG. 16C) per data point.

FIGS. 17A-17D: Quantitative determination of CD37, CD46, CD55 and CD59expression levels on (FIG. 17A) CLL, (FIG. 17B) FL, (FIG. 17C) MCL or(FIG. 17D) DLBCL tumor cells. Expression levels on tumor cells weredetermined by flow cytometry. Antigen quantity is shown as antibodybinding capacity.

FIGS. 18A-18C: CDC mediated by a bispecific CD37 antibody with an Fc-Fcinteraction enhancing mutation on primary tumor cells of patients withCLL, FL, MCL, DLBCL or B-NHL (not further specified). The capacity ofbsIgG1-016-H5L2-LC90S-F405Lx010-H5L2-K409R-E430G to induce CDC on tumorcells derived from patients with (FIG. 18A) CLL, (FIG. 18B) FL and (FIG.18C) MCL, DLBCL or B-NHL (not further specified) was determined by flowcytometry. CDC induction is presented as the percentage lysis determinedby the fraction of 7-AAD positive tumor cells, using 100 μg/mL (FIGS.18A and 18B) or 10 μg/mL (FIG. 18C) ofbsIgG1-016-H5L2-LC90S-F405Lx010-H5L2-K409R-E430G.

FIGS. 19A and 19B: Binding of a bispecific CD37 antibody with an Fc-Fcinteraction enhancing mutation to B cells in human or cynomolgus monkeyblood. Binding of Alexa-488 labeledbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G to B cells in(FIG. 19A) human or (FIG. 19B) cynomolgus monkey blood was determined byflow cytometry. Alexa-488 labeled IgG1-b12 was used as a negativecontrol antibody. Data are shown as geometric mean A488 fluorescenceintensity values, for one representative donor/animal. Error bars showvariation within the experiment (duplicate measurements).

FIGS. 20A and 20B: Cytotoxicity of a bispecific CD37 antibody with anFc-Fc interaction enhancing mutation and an FcγR-interaction enhancedmonoclonal CD37 specific antibody to B cells in human or cynomolgusmonkey blood. (FIG. 20A) Cytotoxicity ofbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G andIgG1-CD37-B2-5239D-I332E to B cells in human blood and (FIG. 20B) ofbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G to B cells incynomolgus monkey blood was determined in a whole blood cytotoxicityassay. IgG1-b12 was used as a negative control antibody. Data are shownas % B cell depletion for one representative donor/animal. Error barsshow variation within the experiment (duplicate measurements).

FIGS. 21A-21D: CDC mediated by a bispecific CD37 antibody with an Fc-Fcinteraction enhancing mutation, a CD20-specific antibody or acombination thereof. (FIGS. 21A-21D) The capacity to induce CDC on tumorcells derived from 2 CLL patients ofbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G, ofatumumab or acombination thereof, at indicated concentrations, was determined exvivo. Data are shown as the % of viable B cells.

FIGS. 22A and 22B: Dose-effect relationship for 3 weekly doses ofbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G in the JVM-3model. (FIG. 22A) Tumor growth of JVM-3 xenografts after treatment withdifferent doses ofbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G or isotypecontrol antibody (IgG1-b12). Mean and SEM of each group (n=10) is shownper time point. (FIG. 22B) Tumor size per mouse at day 25. Mean and SEMare indicated per treatment group. Differences were analyzed by MannWhitney test. Statistically significant differences were indicated asfollows: **: p<0.01; ***: p<0.001.

FIGS. 23A and 23B: Dose-effect relationship for 3 weekly doses ofbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G in the Daudi-lucmodel. (FIG. 23A) Tumor growth (measured by luciferase activity,bioluminescence) of Daudi-luc xenografts after treatment with differentdoses of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G orisotype control antibody (IgG1-b12). Mean and SEM of each group (n=9) isshown per time point. (FIG. 23B) Luciferase activity per mouse at day36. Mean and SEM are indicated per treatment group. Differences wereanalyzed by One Way Anova, Uncorrected Fisher's LSD. Statisticallysignificant differences were indicated as follows: **: p<0.01; ***:p<0.001.

FIGS. 24A-24D: Plasma concentrations ofbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G and IgG1-b12following intravenous injection in SCID mice.

SCID mice were injected with a single i.v. dose of (FIGS. 24A and 24B)100 μg (5 mg/kg) or (FIGS. 24C and 24D) 500 μg (25 mg/kg) ofbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G or IgG1-b12.

FIG. 25. Analysis of binding of CD37 antibodies to CD37 variants withalanine mutations in the extracellular domains. Zscore (fold change) wasdefined as (normalized gMFI[aa position]-μ)/σ, where μ and σ are themean and standard deviation (SD) of the normalized gMFI of all mutants.Residues where the where the zscore was lower than −1.5 (indicated bythe dotted line) were considered ‘loss of binding mutants’. Number abovethe x-axis refer to amino acid positions. Note that x-axis isnon-continuous: the left part (until the striped line) of the axisrepresents aa residues in the small extracellular loop of human CD37which are not alanines or cysteines; the right part of the axisrepresents aa residues in the large extracellular loop of human CD37which are not alanines or cysteines. The dotted line indicates a zscore(fold change) of −1.5.

FIGS. 26A-26F CDC mediated by mixtures of CD37 antibodies with an Fc-Fcinteraction enhancing mutation plus clinically established CD20 antibodyproducts on Raji cells. CDC-mediated killing of Raji cells (% lysisexpressed as the PI-positive cell fraction as determined by flowcytometry) for antibody concentration dilution series of 1:0, 3:1, 1:1,3:1 and 0:1 antibody mixtures (10 μg/mL final concentration) of CD37antibodies with an Fc-Fc interaction enhancing mutation plus standard ofcare CD20 antibody products MabThera (rituximab), Arzerra (ofatumumab)and Gazyva (obinutuzumab, GA101): (FIG. 26A) mixtures withIgG1-37.3-E430G, (FIG. 26B) mixtures with IgG1-G28.1-E430G, (FIG. 26C)mixtures with IgG1-004-E430G, (FIG. 26D) mixtures with IgG1-005-E430G,(FIG. 26E) mixtures with IgG1-010-E430G and (FIG. 26F) mixtures withIgG1-016-E430G.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term “CD37”, as used herein, refers to Leukocyte Antigen CD37, alsoknown as GP52-40, tetraspanin-26, and TSPAN26, which is a heavilyglycosylated transmembrane protein with four transmembrane domains (TMs)and one small and one large extracellular domain. Homo sapiens, i.e.,human, CD37 protein is encoded by a nucleic acid sequence encoding theamino acid sequence shown in SEQ ID NO: 62 (human CD37 protein:UniprotKB/Swissprot P11049). In this amino acid sequence, residues 112to 241 correspond to the large extracellular domain, residues 39 to 59to the small extracellular domain, while the remaining residuescorrespond to transmembrane and cytoplasmic domains. Macacafascicularis, i.e., cynomolgus monkey, CD37 protein is encoded by anucleic acid sequence encoding the amino acid sequence shown in SEQ IDNO: 63 (cynomolgus CD37 protein: Genbank accession no. XP_005589942).Unless contradicted by context the term “CD37” means “human CD37”. Theterm “CD37” includes any variants, isoforms and species homologs of CD37which are naturally expressed by cells, including tumor cells, or areexpressed on cells transfected with the CD37 gene or cDNA.

The term “human CD20” or “CD20” refers to human CD20(UniProtKB/Swiss-Prot No P11836) and includes any variants, isoforms andspecies homologs of CD20 which are naturally expressed by cells,including tumor cells, or are expressed on cells transfected with theCD20 gene or cDNA. Species homologs include rhesus monkey CD20 (Macacamulatta; UniProtKB/Swiss-Prot No H9YXP1) and cynomolgus monkey CD20(Macaca fascicularis).

The term “antibody binding CD37”, “anti-CD37 antibody”, “CD37-bindingantibody”, “CD37-specific antibody”, “CD37 antibody” which may be usedinterchangeably herein, refers to any antibody binding an epitope on theextracellular part of CD37.

The term “antibody” (Ab) in the context of the present invention refersto an immunoglobulin molecule, a fragment of an immunoglobulin molecule,or a derivative of either thereof, which has the ability to specificallybind to an antigen under typical physiological conditions with ahalf-life of significant periods of time, such as at least about 30minutes, at least about 45 minutes, at least about one hour, at leastabout two hours, at least about four hours, at least about 8 hours, atleast about 12 hours, about 24 hours or more, about 48 hours or more,about 3, 4, 5, 6, 7 or more days, etc., or any other relevantfunctionally-defined period (such as a time sufficient to induce,promote, enhance, and/or modulate a physiological response associatedwith antibody binding to the antigen and/or time sufficient for theantibody to recruit an effector activity). The variable regions of theheavy and light chains of the immunoglobulin molecule contain a bindingdomain that interacts with an antigen. The constant regions of theantibodies (Abs) may mediate the binding of the immunoglobulin to hosttissues or factors, including various cells of the immune system (suchas effector cells) and components of the complement system such as C1q,the first component in the classical pathway of complement activation.As indicated above, the term antibody herein, unless otherwise stated orclearly contradicted by context, includes fragments of an antibody thatare antigen-binding fragments, i.e., retain the ability to specificallybind to the antigen. It has been shown that the antigen-binding functionof an antibody may be performed by fragments of a full-length antibody.Examples of antigen-binding fragments encompassed within the term“antibody” include (i) a Fab′ or Fab fragment, a monovalent fragmentconsisting of the V_(L), V_(H), C_(L) and C_(H)1 domains, or amonovalent antibody as described in WO2007059782 (Genmab); (ii) F(ab′)2fragments, bivalent fragments comprising two Fab fragments linked by adisulfide bridge at the hinge region; (iii) a Fd fragment consistingessentially of the V_(H) and C_(H)1 domains; (iv) a Fv fragmentconsisting essentially of the V_(L) and V_(H) domains of a single arm ofan antibody, (v) a dAb fragment (Ward et al., Nature 341, 544-546(1989)), which consists essentially of a V_(H) domain and also calleddomain antibodies (Holt et al; Trends Biotechnol. 2003 November;21(11):484-90); (vi) camelid or nanobodies (Revets et al; Expert OpinBiol Ther. 2005 January; 5(1):111-24) and (vii) an isolatedcomplementarity determining region (CDR). Furthermore, although the twodomains of the Fv fragment, V_(L) and V_(H), are coded for by separategenes, they may be joined, using recombinant methods, by a syntheticlinker that enables them to be made as a single protein chain in whichthe V_(L) and V_(H) regions pair to form monovalent molecules (known assingle chain antibodies or single chain Fv (scFv), see for instance Birdet al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85,5879-5883 (1988)). Such single chain antibodies are encompassed withinthe term antibody unless otherwise noted or clearly indicated bycontext. Although such fragments are generally included within themeaning of antibody, they collectively and each independently are uniquefeatures of the present invention, exhibiting different biologicalproperties and utility. These and other useful antibody fragments in thecontext of the present invention, as well as bispecific formats of suchfragments, are discussed further herein. For the bispecific antibodiesof the invention such fragments are linked to an Fc domain. It alsoshould be understood that the term antibody, unless specified otherwise,also includes polyclonal antibodies, monoclonal antibodies (mAbs),antibody-like polypeptides, such as chimeric antibodies and humanizedantibodies, and antibody fragments retaining the ability to specificallybind to the antigen (antigen-binding fragments) provided by any knowntechnique, such as enzymatic cleavage, peptide synthesis, andrecombinant techniques. An antibody as generated can possess anyisotype.

The term “bispecific antibody” refers to antibody having specificitiesfor at least two different, typically non-overlapping, epitopes. Suchepitopes may be on the same or different targets. For the presentinvention the epitopes are on the same target, namely CD37. Examples ofdifferent classes of bispecific antibodies comprising an Fc regioninclude but are not limited to: asymmetric bispecific molecules, e.g.,IgG-like molecules with complementary CH3 domains; and symmetricbispecific molecules, e.g., recombinant IgG-like dual targetingmolecules wherein each antigen-binding region of the molecule binds atleast two different epitopes.

Examples of bispecific molecules include but are not limited to Triomab®(Trion Pharma/Fresenius Biotech, WO/2002/020039), Knobs-into-Holes(Genentech, WO 1998/50431), CrossMAbs (Roche, WO 2009/080251, WO2009/080252, WO 2009/080253), electrostatically-matched Fc-heterodimericmolecules (Amgen, EP1870459 and WO2009089004; Chugai, US201000155133;Oncomed, WO 2010/129304), LUZ-Y (Genentech), DIG-body, PIG-body andTIG-body (Pharmabcine), Strand Exchange Engineered Domain body(SEEDbody) (EMD Serono, WO2007110205), Bispecific IgG1 and IgG2(Pfizer/Rinat, WO 2011/143545), Azymetric scaffold (Zymeworks/Merck,WO2012058768), mAb-Fv (Xencor, WO 2011/028952), XmAb (Xencor), Bivalentbispecific antibodies (Roche, WO 2009/080254), Bispecific IgG (EliLilly), DuoBody® molecules (Genmab A/S, WO 2011/131746), DuetMab(Medimmune, US2014/0348839), BicIonics (Merus, WO 2013/157953),NovImmune (KABodies, WO 2012/023053), FcΔAdp (Regeneron, WO2010/151792), (DT)-Ig (GSK/Domantis), Two-in-one Antibody or Dual ActionFabs (Genentech, Adimab), mAb2 (F-Star, WO 2008/003116), Zybody™molecules (Zyngenia), CovX-body (CovX/Pfizer), FynomAbs (Covagen/JanssenCilag), DutaMab (Dutalys/Roche), iMab (MedImmune), Dual Variable Domain(DVD)-Ig™ (Abbott), dual domain double head antibodies (Unilever; SanofiAventis, WO 2010/0226923), Ts2Ab (MedImmune/AZ), BsAb (Zymogenetics),HERCULES (Biogen Idec, U.S. Pat. No. 7,951,918), scFv-fusions(Genentech/Roche, Novartis, Immunomedics, Changzhou Adam Biotech Inc, CN102250246), TvAb (Roche, WO2012/025525, WO2012/025530), ScFv/Fc Fusions,SCORPION (Emergent BioSolutions/Trubion, Zymogenetics/BMS), Interceptor(Emergent), Dual Affinity Retargeting Technology (Fc-DART™)(MacroGenics, WO2008/157379, WO2010/080538), BEAT (Glenmark), Di-Diabody(Imclone/Eli Lilly) and chemically crosslinked mAbs (Karmanos CancerCenter), and covalently fused mAbs (AIMM therapeutics).

The term “full-length antibody”, as used herein, refers to an antibody(e.g., a parent or variant antibody) which contains all heavy and lightchain constant and variable domains corresponding to those that arenormally found in a wild-type antibody of that class or isotype.

The term “chimeric antibody” as used herein, refers to an antibodywherein the variable region is derived from a non-human species (e.g.derived from rodents) and the constant region is derived from adifferent species, such as human. Chimeric antibodies may be generatedby antibody engineering. “Antibody engineering” is a term used genericfor different kinds of modifications of antibodies, and which is awell-known process for the skilled person. In particular, a chimericantibody may be generated by using standard DNA techniques as describedin Sambrook et al., 1989, Molecular Cloning: A laboratory Manual, NewYork: Cold Spring Harbor Laboratory Press, Ch. 15. Thus, the chimericantibody may be a genetically or an enzymatically engineered recombinantantibody. It is within the knowledge of the skilled person to generate achimeric antibody, and thus, generation of the chimeric antibodyaccording to the present invention may be performed by other methodsthan described herein. Chimeric monoclonal antibodies for therapeuticapplications are developed to reduce antibody immunogenicity. They maytypically contain non-human (e.g. murine) variable regions, which arespecific for the antigen of interest, and human constant antibody heavyand light chain domains. The terms “variable region” or “variabledomains” as used in the context of chimeric antibodies, refers to aregion which comprises the CDRs and framework regions of both the heavyand light chains of the immunoglobulin.

The term “oligomer”, as used herein, refers to a molecule that consistsof more than one but a limited number of monomer units (e.g. antibodies)in contrast to a polymer that, at least in principle, consists of anunlimited number of monomers. Exemplary oligomers are dimers, trimers,tetramers, pentamers and hexamers. Likewise, “oligomerization” such ase.g. “hexamerization”, as used herein, means that there is an increasein the distribution of antibodies and/or other dimeric proteinscomprising target-binding regions according to the invention intooligomers, such as hexamers. The increased formation of oligomers suchas hexamers is due to increased Fc-Fc interaction after binding tomembrane-bound targets.

The term “antigen-binding region”, “antigen binding region”, “bindingregion” or antigen binding domain, as used herein, refers to a region ofan antibody which is capable of binding to the antigen. This bindingregion is typically defined by the VH and VL domains of the antibodywhich may be further subdivided into regions of hypervariability (orhypervariable regions which may be hypervariable in sequence and/or formof structurally defined loops), also termed complementarity determiningregions (CDRs), interspersed with regions that are more conserved,termed framework regions (FRs). The antigen can be any molecule, such asa polypeptide, e.g. present on a cell, bacterium, or virion or insolution. The terms “antigen” and “target” may, unless contradicted bythe context, be used interchangeably in the context of the presentinvention.

The term “target”, as used herein, refers to a molecule to which theantigen binding region of the antibody binds. The target includes anyantigen towards which the raised antibody is directed. The term“antigen” and “target” may in relation to an antibody be usedinterchangeably and constitute the same meaning and purpose with respectto any aspect or embodiment of the present invention.

The term “humanized antibody” as used herein, refers to a geneticallyengineered non-human antibody, which contains human antibody constantdomains and non-human variable domains modified to contain a high levelof sequence homology to human variable domains. This can be achieved bygrafting of the six non-human antibody complementarity-determiningregions (CDRs), which together form the antigen binding site, onto ahomologous human acceptor framework region (FR) (see WO92/22653 andEP0629240). In order to fully reconstitute the binding affinity andspecificity of the parental antibody, the substitution of frameworkresidues from the parental antibody (i.e. the non-human antibody) intothe human framework regions (back-mutations) may be required. Structuralhomology modeling may help to identify the amino acid residues in theframework regions that are important for the binding properties of theantibody. Thus, a humanized antibody may comprise non-human CDRsequences, primarily human framework regions optionally comprising oneor more amino acid back-mutations to the non-human amino acid sequence,and fully human constant regions. Optionally, additional amino acidmodifications, which are not necessarily back-mutations, may be appliedto obtain a humanized antibody with preferred characteristics, such asaffinity and biochemical properties.

Humanized antibodies can be generated using immunized rabbits,humanization of rabbit antibodies using germline humanization(CDR-grafting) technology, and, if necessary, by back-mutating residueswhich may be critical for the antibody binding properties, as identifiedin structural modeling, to rabbit residues. Screening for potential Tcell epitopes can be applied.

The term “human antibody” as used herein, refers to antibodies havingvariable and constant regions derived from human germline immunoglobulinsequences. Human antibodies may include amino acid residues not encodedby human germline immunoglobulin sequences (e.g., mutations introducedby random or site-specific mutagenesis in vitro or by somatic mutationin vivo). However, the term “human antibody”, as used herein, is notintended to include antibodies in which CDR sequences derived from thegermline of another mammalian species, such as a mouse, have beengrafted onto human framework sequences. Human monoclonal antibodies ofthe invention can be produced by a variety of techniques, includingconventional monoclonal antibody methodology, e.g., the standard somaticcell hybridization technique of Kohler and Milstein, Nature 256: 495(1975). Although somatic cell hybridization procedures are preferred, inprinciple, other techniques for producing monoclonal antibody can beemployed, e.g., viral or oncogenic transformation of B-lymphocytes orphage display techniques using libraries of human antibody genes.

A suitable animal system for preparing hybridomas that secrete humanmonoclonal antibodies is the murine system. Hybridoma production in themouse is a very well established procedure. Immunization protocols andtechniques for isolation of immunized splenocytes for fusion are knownin the art. Fusion partners (e.g., murine myeloma cells) and fusionprocedures are also known.

Human monoclonal antibodies can be generated using e.g. transgenic ortranschromosomal mice or rabbits carrying parts of the human immunesystem rather than the mouse or rabbit system.

The term “immunoglobulin” refers to a class of structurally relatedglycoproteins consisting of two pairs of polypeptide chains, one pair oflight (L) low molecular weight chains and one pair of heavy (H) chains,all four inter-connected by disulfide bonds. The structure ofimmunoglobulins has been well characterized. See for instanceFundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989)). Briefly, each heavy chain typically is comprised of a heavychain variable region (abbreviated herein as V_(H) or VH) and a heavychain constant region (abbreviated herein as C_(H) or CH). The heavychain constant region typically is comprised of three domains, C_(H)1,C_(H)2, and C_(H)3. Each light chain typically is comprised of a lightchain variable region (abbreviated herein as V_(L) or VL) and a lightchain constant region (abbreviated herein as C_(L) or CL). The lightchain constant region typically is comprised of one domain, C_(L). TheV_(H) and V_(L) regions may be further subdivided into regions ofhypervariability (or hypervariable regions which may be hypervariable insequence and/or form of structurally defined loops), also termedcomplementarity determining regions (CDRs), interspersed with regionsthat are more conserved, termed framework regions (FRs). Each V_(H) andV_(L) is typically composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196,901-917 (1987)). Unless otherwise stated or contradicted by context, CDRsequences herein are identified according to IMGT rules (Brochet X.,Nucl Acids Res. 2008; 36:W503-508 and Lefranc M P., Nucleic AcidsResearch 1999; 27:209-212; see also internet http addresshttp://www.imgt.org/). Unless otherwise stated or contradicted bycontext, reference to amino acid positions in the constant regions inthe present invention is according to the EU-numbering (Edelman et al.,Proc Natl Acad Sci USA. 1969 May; 63(1):78-85; Kabat et al., Sequencesof Proteins of Immunological Interest, Fifth Edition. 1991 NIHPublication No. 91-3242).

When used herein, unless contradicted by context, the term “Fab-arm” or“arm” refers to one heavy chain-light chain pair and is usedinterchangeably with “half molecules” herein. Accordingly, a “Fab-arm”comprises the variable regions of the heavy chain and light chain aswell as the constant region of the light chain and the constant regionof the heavy chain which comprises the CH1 region, the hinge, the CH2region and the CH3 region of an immunoglobulin. The “CH1 region” referse.g. to the region of a human IgG1 antibody corresponding to amino acids118-215 according to the EU numbering. Thus, the Fab fragment comprisesthe binding region of an immunoglobulin.

The term “fragment crystallizable region”, “Fc region”, “Fc fragment” or“Fc domain”, which may be used interchangeably herein, refers to anantibody region comprising, arranged from amino-terminus tocarboxy-terminus, at least a hinge region, a CH2 domain and a CH3domain. An Fc region of an IgG1 antibody can, for example, be generatedby digestion of an IgG1 antibody with papain. The Fc region of anantibody may mediate the binding of the immunoglobulin to host tissuesor factors, including various cells of the immune system (such aseffector cells) and components of the complement system such as C1q, thefirst component in the classical pathway of complement activation. Theterm “hinge region”, as used herein, is intended to refer to the hingeregion of an immunoglobulin heavy chain. Thus, for example the hingeregion of a human IgG1 antibody corresponds to amino acids 216-230according to the EU numbering.

The term “core hinge” or “core hinge region” as used herein refers tothe four amino acids corresponding to positions 226-229 of a human IgG1antibody.

The term “CH2 region” or “CH2 domain”, as used herein, is intended torefer the CH2 region of an immunoglobulin heavy chain. Thus, for examplethe CH2 region of a human IgG1 antibody corresponds to amino acids231-340 according to the EU numbering. However, the CH2 region may alsobe any of the other isotypes or allotypes as described herein.

The term “CH3 region” or “CH3 domain” as used herein, is intended torefer to the CH3 region of an immunoglobulin heavy chain. Thus, forexample the CH3 region of a human IgG1 antibody corresponds to aminoacids 341-447 according to the EU numbering. However, the CH3 region mayalso be any of the other isotypes or allotypes as described herein.

As used herein, the term “isotype” refers to the immunoglobulin class(for instance IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) that isencoded by heavy chain constant region genes.

The term “monovalent antibody” means in the context of the presentinvention that an antibody molecule is capable of binding a singlemolecule of the antigen, and thus is not capable of antigencrosslinking.

A “CD37 antibody” or “anti-CD37 antibody” is an antibody as describedabove, which binds specifically to the antigen CD37.

A “CD37×CD37 antibody” or “anti-CD37×CD37 antibody” is a bispecificantibody, which comprises two different antigen-binding regions, one ofwhich binds specifically to a first epitope on the antigen CD37 and asecond which binds specifically to a different epitope on CD37.

In an embodiment, the bispecific antibody of the invention is isolated.An “isolated bispecific antibody,” as used herein, is intended to referto a bispecific antibody which is substantially free of other antibodieshaving different antigenic specificities (for instance an isolatedbispecific antibody that specifically binds to CD37 is substantiallyfree of monospecific antibodies that specifically bind to CD37).

The term “epitope” means a protein determinant capable of binding to anantigen-binding region of an antibody (“paratope”). Epitopes usuallyconsist of surface groupings of molecules such as amino acids or sugarside chains and usually have specific three-dimensional structuralcharacteristics, as well as specific charge characteristics.Conformational and nonconformational epitopes are distinguished in thatthe binding to the former but not the latter is lost in the presence ofdenaturing solvents. Epitope mapping techniques can determine“structural epitopes” or “functional epitopes”. Structural epitopes aredefined as those residues within a structure that are in direct contactwith the antibody and can for example be assessed by structure basedmethods such as X-ray crystallography. A structural epitope may compriseamino acid residues directly involved in the binding of an antibody aswell as other amino acid residues, which are not directly involved inthe binding, such as amino acid residues which are effectively blockedor covered by antibody (in other words, the amino acid residue is withinthe footprint of the antibody). Functional epitope are defined as thoseresidues that make energetic contributions to the antigen-antibodybinding interaction and can for example be assessed by site-directedmutagenesis such as alanine scanning (Cunningham, B. C., & Wells, J. A.(1993) Journal of Molecular Biology; Clackson, T., & Wells, J. (1995)Science, 267(5196), 383-386). A functional epitope may comprise aminoacid residues directly involved in the binding of an antibody as well asother amino acid residues which are not directly involved in thebinding, such as amino acid residues which cause conformational changesto the location of residues involved in direct interactions (Greenspan,N. S., & Di Cera, E. (1999) Nature Biotechnology, 17(10), 936-937). Incase of antibody-antigen interactions, the functional epitope may beused to distinguish antibody molecules between each other. A functionalepitope may be determined by use of the method of alanine scanning asdescribed in Example 17. Thus, amino acids in the protein may besubstituted with alanines thereby generating a series of mutantproteins, binding of the antigen-binding region of the antibody to themutant protein is reduced as compared to a wild type protein; reducedbinding being determined as standardized log(fold change) (expressed asz-scores) in binding of said antibody being less than −1.5 as set forthin Example 17.

The term “monoclonal antibody” as used herein refers to a preparation ofantibody molecules essentially of single molecular composition. Amonoclonal antibody composition displays a single binding specificityand affinity for a particular epitope. Accordingly, the term “humanmonoclonal antibody” refers to antibodies displaying a single bindingspecificity which have variable and constant regions derived from humangermline immunoglobulin sequences. The human monoclonal antibodies maybe generated by a hybridoma which includes a B cell obtained from atransgenic or transchromosomal non-human animal, such as a transgenicmouse, having a genome comprising a human heavy chain transgene and alight chain transgene, fused to an immortalized cell.

As used herein, the term “binding” in the context of the binding of anantibody to a predetermined antigen typically is a binding with anaffinity corresponding to a K_(D) of about 10⁻⁶ M or less, e.g. 10⁻⁷ Mor less, such as about 10⁻⁸ M or less, such as about 10⁻⁹ M or less,about 10⁻¹⁰ M or less, or about 10⁻¹¹M or even less when determined byfor instance BioLayer Interferometry (BLI) technology in a Octet HTXinstrument using the antibody as the ligand and the antigen as theanalyte, and wherein the antibody binds to the predetermined antigenwith an affinity corresponding to a K_(D) that is at least ten-foldlower, such as at least 100-fold lower, for instance at least 1,000-foldlower, such as at least 10,000-fold lower, for instance at least100,000-fold lower than its K_(D) of binding to a non-specific antigen(e.g., BSA, casein) other than the predetermined antigen or a closelyrelated antigen. The amount with which the K_(D) of binding is lower isdependent on the K_(D) of the antibody, so that when the K_(D) of theantibody is very low, then the amount with which the K_(D) of binding tothe antigen is lower than the K_(D) of binding to a non-specific antigenmay be at least 10,000-fold (that is, the antibody is highly specific).

The term “K_(D)” (M), as used herein, refers to the dissociationequilibrium constant of a particular antibody-antigen interaction.

“Affinity”, as used herein, and “K_(D)” are inversely related, that is,higher affinity is intended to refer to lower K_(D), and lower affinityis intended to refer to higher K_(D).

As used herein, an antibody which “competes” or “cross-competes” is usedinterchangeably with an antibody which “blocks” or “cross-blocks” withanother antibody, i.e. a reference antibody, and means that the antibodyand the reference antibody compete for binding to human CD37, e.g. asdetermined in the assay described in Examples 7 herein. In oneembodiment the antibody binds with less than 50%, such as less than 20%,such as less than 15% of its maximum binding in the presence of thecompeting reference antibody.

As used herein, an antibody which “does not compete” or “does notcross-compete” or “does not block” with another antibody, i.e. areference antibody, means that the antibody and the reference antibodydo not compete for binding to human CD37, e.g. as determined in theassay described in Examples 7 herein. For some pairs of antibody andreference antibody, non-competition in the assay of Example 7 is onlyobserved when one antibody is bound to an antigen on a cell and theother is used to compete, and not vice versa. The term “does not competewith” or “non-competition” or “non-blocking” when used herein is alsointended to cover such combinations of antibodies. In one embodiment theantibody binds with at least 75%, such as least 80%, such as at least85% of its maximum binding in the presence of the reference antibody.

The term “Fc-Fc interaction enhancing mutation”, as used herein, refersto a mutation in IgG antibodies that strengthens Fc-Fc interactionsbetween neighboring IgG antibodies that are bound to a cell surfacetarget. This may result in enhanced oligomer formation such as e.g.hexamerization of the target-bound antibodies, while the antibodymolecules remain monomeric in solution as described in WO 2013/004842;WO 2014/108198 both which are hereby incorporated by reference.

The term “Fc effector functions” or “Fc-mediated effector functions” asused herein, is intended to refer to functions that are a consequence ofbinding a polypeptide or antibody to its target, such as an antigen, ona cell membrane, and subsequent interaction of the IgG Fc domain withmolecules of the innate immune system (e.g. soluble molecules ormembrane-bound molecules). Examples of Fc effector functions include (i)C1q-binding, (ii) complement activation, (iii) complement-dependentcytotoxicity (CDC), (iv) antibody-dependent cell-mediated cytotoxicity(ADCC), (v) Fc-gamma receptor-binding, (vi) antibody-dependent cellularphagocytosis (ADCP), (vii) complement-dependent cellular cytotoxicity(CDCC), (viii) complement-enhanced cytotoxicity, (ix) binding tocomplement receptor of an opsonized antibody mediated by the antibody,(x) opsonisation, and (xi) a combination of any of (i) to (x).

When used herein the term “heterodimeric interaction between the firstand second CH3 regions” refers to the interaction between the first CH3region of the first Fc-region and the second CH3 region of the secondFc-region in a first-CH3/second-CH3 heterodimeric protein. A bispecificantibody is an example of a heterodimeric protein.

When used herein the term “homodimeric interactions of the first andsecond CH3 regions” refers to the interaction between a first CH3 regionand another first CH3 region in a first-CH3/first-CH3 homodimericprotein and the interaction between a second CH3 region and anothersecond CH3 region in a second-CH3/second-CH3 homodimeric protein. Amonoclonal antibody is an example of a homodimeric protein.

The term “reducing conditions” or “reducing environment” refers to acondition or an environment in which a substrate, such as e.g. acysteine residue in the hinge region of an antibody, is more likely tobecome reduced than oxidized.

The present invention also provides bispecific antibodies comprisingfunctional variants of the V_(L) regions, V_(H) regions, or one or moreCDRs of the bispecific antibodies of the examples. A functional variantof a V_(L), V_(H), or CDR used in the context of a bispecific antibodystill allows each arm of the bispecific antibody to retain at least asubstantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% ormore) of the affinity and/or the specificity/selectivity of the parentbispecific antibody and in some cases such a bispecific antibody may beassociated with greater affinity, selectivity and/or specificity thanthe parent bispecific antibody. Such functional variants typicallyretain significant sequence identity to the parent bispecific antibody.The percent identity between two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The percent identity betweentwo nucleotide or amino acid sequences may e.g. be determined using thealgorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17(1988) which has been incorporated into the ALIGN program (version 2.0),using a PAM120 weight residue table, a gap length penalty of 12 and agap penalty of 4. In addition, the percent identity between two aminoacid sequences may be determined using the Needleman and Wunsch, J. Mol.Biol. 48, 444-453 (1970) algorithm.

Exemplary variants include those which differ from VH and/or VL and/orCDR regions of the parent bispecific antibody sequences mainly byconservative substitutions; for instance 10, such as 9, 8, 7, 6, 5, 4,3, 2 or 1 of the substitutions in the variant are conservative aminoacid residue replacements. Preferably, a variant contains at most 10amino acid substitutions in the VH and/or VL region of the parentantibody, such as at most 9, 8, 7, 6, 5, 4, 3, 2 or at most 1 amino acidsubstitution. Preferably such substitutions are conservativesubstitutions especially so if the substitutions are in a CDR sequence.

In the context of the present invention, conservative substitutions maybe defined by substitutions within the classes of amino acids reflectedin the following table:

Amino acid residue classes for conservative substitutions AcidicResidues Asp (D) and Glu (E) Basic Residues Lys (K), Arg (R), and His(H) Hydrophilic Uncharged Residues Ser (S), Thr (T), Asn (N), and Gln(Q) Aliphatic Uncharged Residues Gly (G), Ala (A), Val (V), Leu (L), andIle (I) Non-polar Uncharged Residues Cys (C), Met (M), and Pro (P)Aromatic Residues Phe (F), Tyr (Y), and Trp (W)

In the context of the present invention, the following notations are,unless otherwise indicated, used to describe a mutation; i) substitutionof an amino acid in a given position is written as e.g. K409R whichmeans a substitution of a Lysine in position 409 with an Arginine; andii) for specific variants the specific three or one letter codes areused, including the codes Xaa and X to indicate any amino acid residue.Thus, the substitution of Lysine with Arginine in position 409 isdesignated as: K409R, and the substitution of Lysine with any amino acidresidue in position 409 is designated as K409X. In case of deletion ofLysine in position 409 it is indicated by K409*.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which an expression vectorhas been introduced, e.g. an expression vector encoding an antibody ofthe invention. Recombinant host cells include, for example,transfectomas, such as CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F,PER.C6 or NS0 cells, and lymphocytic cells.

The term “treatment” refers to the administration of an effective amountof a therapeutically active bispecific antibody of the present inventionwith the purpose of easing, ameliorating, arresting or eradicating(curing) symptoms or disease states.

The term “effective amount” or “therapeutically effective amount” refersto an amount effective, at dosages and for periods of time necessary, toachieve a desired therapeutic result. A therapeutically effective amountof a bispecific antibody may vary according to factors such as thedisease state, age, sex, and weight of the individual, and the abilityof the bispecific antibody to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the antibody or antibody portion areoutweighed by the therapeutically beneficial effects.

The term “anti-idiotypic antibody” refers to an antibody whichrecognizes unique determinants generally associated with theantigen-binding site of an antibody.

Embodiments of the Invention

In a first main embodiment the invention provides a bispecific antibodycomprising a first and second antigen binding region binding to humanCD37 having the sequence of SEQ ID NO: 62, and a first and second Fcregion of a human immunoglobulin, wherein the first and second antigenbinding regions bind different epitopes on CD37 and wherein the firstand second Fc regions comprises one or more amino acid mutations whichmutation(s) enhances the Fc-Fc interaction between the bispecificantibodies upon binding to membrane-bound targets compared to the Fc-Fcinteraction between bispecific antibodies not having said mutation(s).Hereby a bispecific anti-CD37 antibody is provided which binds twodifferent epitopes on CD37. Preferably the two epitopes are such thatboth binding arms can bind the same protein and thus such that eachbinding arm does not block binding of the other arm and/or does notcompete for binding with the other binding arm of the bispecificmolecule. Also, the bispecific antibody comprises a mutation thatenhances the Fc-Fc interaction between two or more of the bispecificantibodies of the invention. This has the effect that the bispecificmolecules form oligomers upon binding to CD37 expressed on the plasmamembrane of the target cell. The Fc-Fc interaction is enhanced comparedto a molecule that is identical except for the mutation. Preferably themutation is in the Fc region of the bispecific molecule. In oneembodiment it is a single amino acid substitution in the Fc region ofthe bispecific molecule. It is preferably a symmetric substitutionmeaning that both half molecules (parental antibodies) have themutation. It is a further advantage of the present bispecific antibodythat it has enhanced CDC and/or ADCC effector functions compared to anidentical bispecific molecule not having the Fc-Fc interaction enhancingmutation. Surprisingly the bispecific molecule also has improved CDCand/or ADCC compared to a combination of the two parental monoclonalanti-CD37 antibodies which are mutated to have enhanced Fc-Fcinteractions, and improved CDC and/or ADCC compared to either parentalmonoclonal anti-CD37 antibody which is mutated to have enhanced Fc-Fcinteractions by itself. Thus, the bispecific antibody of the inventionis more potent in inducing CDC and/or ADCC than a combination of anantibody having the first antigen binding region and a second antibodyhaving the second antigen binding region and where both antibodiescomprise the Fc-Fc interaction enhancing mutation, or compared to thesingle monoclonal anti-CD37 antibodies having the first or the secondantigen binding regions and which comprise the Fc-Fc interactionenhancing mutation.

In an embodiment of the invention the first antigen binding region ofthe bispecific antibody is obtained from an antibody which competes forbinding to human CD37 with a CD37 antibody comprising the CDR sequences:

-   -   VH CDR1 sequence set forth in SEQ ID NO: 16,    -   VH CDR2 sequence set forth in SEQ ID NO: 17,    -   VH CDR3 sequence set forth in SEQ ID NO: 18,    -   VL CDR1 sequence set forth in SEQ ID NO: 20,    -   VL CDR2 sequence KAS, and    -   VL CDR3 sequence set forth in SEQ ID NO: 21. [010]

Preferably competition for binding is determined according to example 7.

In another embodiment the first antigen binding region of the bispecificantibody binds to the same epitope on human CD37 as a CD37 antibodycomprising the CDR sequences:

-   -   VH CDR1 sequence set forth in SEQ ID NO: 16,    -   VH CDR2 sequence set forth in SEQ ID NO: 17,    -   VH CDR3 sequence set forth in SEQ ID NO: 18,    -   VL CDR1 sequence set forth in SEQ ID NO: 20,    -   VL CDR2 sequence KAS, and    -   VL CDR3 sequence set forth in SEQ ID NO: 21. [010]

In a further embodiment of the invention the first antigen bindingregion of the bispecific antibody comprises the CDR sequences:

-   -   VH CDR1 sequence set forth in SEQ ID NO: 16,    -   VH CDR2 sequence set forth in SEQ ID NO: 17,    -   VH CDR3 sequence set forth in SEQ ID NO: 18,    -   VL CDR1 sequence set forth in SEQ ID NO: 20,    -   VL CDR2 sequence KAS, and    -   VL CDR3 sequence set forth in SEQ ID NO: 21. [010]

In a further embodiment of the invention the first antigen bindingregion of the bispecific antibody of the invention comprise the VH andVL sequences:

-   -   (i) VH sequence set forth in SEQ ID NO: 15 and VL sequence set        forth in SEQ ID NO: 19 or    -   (ii) VH sequence having at least 90% identity, such as at least        95% identity, such as at least 98% identity, such as at least        99% identity and a VL sequence having at least 90% identity,        such as at least 95% identity, such as at least 98% identity,        such as at least 99% identity with the VH sequence and VL        sequences of SEQ ID Nos 15 and 19.

In a further embodiment of the invention the first antigen bindingregion of the bispecific antibody is obtained from an antibody whichcompetes for binding to human CD37 with a CD37 antibody comprising theCDR sequences:

-   -   VH CDR1 sequence set forth in SEQ ID NO: 9,    -   VH CDR2 sequence set forth in SEQ ID NO: 10,    -   VH CDR3 sequence set forth in SEQ ID NO: 11,    -   VL CDR1 sequence set forth in SEQ ID NO: 13,    -   VL CDR2 sequence: AAS, and    -   VL CDR3 sequence set forth in SEQ ID NO: 14. [005]

In a further embodiment of the invention the first antigen bindingregion of the bispecific antibody binds to the same epitope on humanCD37 as a CD37 antibody comprising the CDR sequences:

-   -   VH CDR1 sequence set forth in SEQ ID NO: 9,    -   VH CDR2 sequence set forth in SEQ ID NO: 10,    -   VH CDR3 sequence set forth in SEQ ID NO: 11,    -   VL CDR1 sequence set forth in SEQ ID NO: 13,    -   VL CDR2 sequence: AAS, and    -   VL CDR3 sequence set forth in SEQ ID NO: 14. [005]

In one embodiment of the invention the first antigen binding region ofthe bispecific antibody has a functional epitope comprising one or moreof the amino acids Y182, D189, T191, I192, D194, K195, V196, I197 andP199 of SEQ ID No: 62 (CD37).

In one embodiment of the invention said first antigen binding regionbinds to a functional epitope comprising one or more of the amino acidsselected from the group consisting of: Y182, D189, T191, I192, D194,K195, V196, I197 and P199 of SEQ ID No: 62 (CD37).

In one embodiment of the invention the first antigen binding region ofthe bispecific antibody binds to a functional epitope on CD37, whereinbinding to a mutant CD37 in which any one or more of the amino acidresidues at positions corresponding to positions Y182, D189, T191, I192,D194, K195, V196, I197 and P199 of SEQ ID no 62 (CD37). has/have beensubstituted with alanines, is reduced as compared to wild type CD37having the amino acid sequence set forth in SEQ ID NO: 62; reducedbinding being determined as zscore (fold change) in binding of saidantibody being lowed that −1.5, wherein zscore (fold change) in bindingis calculated as set forth in Example 17.

In a further embodiment of the invention the first antigen bindingregion of the bispecific antibody comprises the CDR sequences:

-   -   VH CDR1 sequence set forth in SEQ ID NO: 9,    -   VH CDR2 sequence set forth in SEQ ID NO: 10,    -   VH CDR3 sequence set forth in SEQ ID NO: 11,    -   VL CDR1 sequence set forth in SEQ ID NO: 13,    -   VL CDR2 sequence: AAS, and    -   VL CDR3 sequence set forth in SEQ ID NO: 14. [005]

In a further embodiment of the invention the first antigen bindingregion of the bispecific antibody comprise the VH and VL sequences:

-   -   (i) VH sequence set forth in SEQ ID NO: 8 and VL sequence set        forth in SEQ ID NO: 12 or    -   (ii) VH sequence having at least 90% identity, such as at least        95% identity, such as at least 98% identity, such as at least        99% identity and a VL sequence having at least 90% identity,        such as at least 95% identity, such as at least 98% identity,        such as at least 99% identity with the VH sequence and VL        sequences of SEQ ID Nos 8 and 12.

In a further embodiment of the invention the second antigen bindingregion of the bispecific antibody is obtained from an antibody whichcompetes for binding to human CD37 with a CD37 antibody comprising theCDR sequences selected from the group comprising:

-   -   (i) VH CDR1 sequence set forth in SEQ ID NO: 23,        -   VH CDR2 sequence set forth in SEQ ID NO: 24,        -   VH CDR3 sequence set forth in SEQ ID NO: 25,        -   VL CDR1 sequence set forth in SEQ ID NO: 27,        -   VL CDR2 sequence: YAS, and        -   VL CDR3 sequence set forth in SEQ ID NO: 28; [016]    -   (ii) VH CDR1 sequence set forth in SEQ ID NO: 2,        -   VH CDR2 sequence set forth in SEQ ID NO: 3,        -   VH CDR3 sequence set forth in SEQ ID NO: 4,        -   VL CDR1 sequence set forth in SEQ ID NO: 6,        -   VL CDR2 sequence: EAS, and        -   VL CDR3 sequence set forth in SEQ ID NO: 7; [004]    -   (iii) VH CDR1 sequence set forth in SEQ ID NO: 40,        -   VH CDR2 sequence set forth in SEQ ID NO: 41,        -   VH CDR3 sequence set forth in SEQ ID NO: 42,        -   VL CDR1 sequence set forth in SEQ ID NO: 44,        -   VL CDR2 sequence: FAK, and        -   VL CDR3 sequence set forth in SEQ ID NO: 45; [G28.1] and    -   (iv) VH CDR1 sequence set forth in SEQ ID NO: 47,        -   VH CDR2 sequence set forth in SEQ ID NO: 48,        -   VH CDR3 sequence set forth in SEQ ID NO: 49,        -   VL CDR1 sequence set forth in SEQ ID NO: 51,        -   VL CDR2 sequence: VAT and        -   VL CDR3 sequence set forth in SEQ ID NO: 52. [37.3]

In a further embodiment of the invention the second antigen bindingregion of the bispecific antibody is obtained from an antibody whichcompetes for binding to human CD37 with a CD37 antibody comprising theCDR sequences selected from the group consisting of:

-   -   (i) VH CDR1 sequence set forth in SEQ ID NO: 23,        -   VH CDR2 sequence set forth in SEQ ID NO: 24,        -   VH CDR3 sequence set forth in SEQ ID NO: 25,        -   VL CDR1 sequence set forth in SEQ ID NO: 27,        -   VL CDR2 sequence: YAS, and        -   VL CDR3 sequence set forth in SEQ ID NO: 28; [016]    -   (ii) VH CDR1 sequence set forth in SEQ ID NO: 2,        -   VH CDR2 sequence set forth in SEQ ID NO: 3,        -   VH CDR3 sequence set forth in SEQ ID NO: 4,        -   VL CDR1 sequence set forth in SEQ ID NO: 6,        -   VL CDR2 sequence: EAS, and        -   VL CDR3 sequence set forth in SEQ ID NO: 7; [004]    -   (iii) VH CDR1 sequence set forth in SEQ ID NO: 40,        -   VH CDR2 sequence set forth in SEQ ID NO: 41,        -   VH CDR3 sequence set forth in SEQ ID NO: 42,        -   VL CDR1 sequence set forth in SEQ ID NO: 44,        -   VL CDR2 sequence: FAK, and        -   VL CDR3 sequence set forth in SEQ ID NO: 45; [G28.1] and    -   (iv) VH CDR1 sequence set forth in SEQ ID NO: 47,        -   VH CDR2 sequence set forth in SEQ ID NO: 48,        -   VH CDR3 sequence set forth in SEQ ID NO: 49,        -   VL CDR1 sequence set forth in SEQ ID NO: 51,        -   VL CDR2 sequence: VAT and        -   VL CDR3 sequence set forth in SEQ ID NO: 52. [37.3]

Hereby bispecific antibodies are provided wherein the first and secondantigen binding regions bind different epitopes on human CD37. Theinventors of the present invention have found that the antibodies havingthe CDR sequences of antibody 005 (SEQ ID NOs 9, 10, 11 and 13, 13a, 14)and antibody 010 (SEQ ID NOs 16, 17, 18 and 20, 20a, 21) compete forbinding to human CD37 and do not compete for binding to CD37 with any ofthe antibodies having the CDR sequences of antibodies 016 (SEQ ID NOs23, 24, 25 and 27, 27a, 28), 004 (SEQ ID NOs 2, 3, 4 and 6, 6a, 7),G28.1 (SEQ ID NOs 40, 41, 42 and 44, 44a, 45) and 37.3 (SEQ ID NOs 47,48, 49 and 51, 51a, 52). The 016, 004, G28.1 and 37.3 antibodies havehowever been found to compete with each other for binding to human CD37.Thus, a bispecific antibody comprising a first binding arm which isobtained from an antibody which competes for binding with either of orboth of the 005 or 010 antibodies and a second binding arm which isobtained from an antibody which competes for binding with any of 016,004, G28.1 and 37.3 or with all of these is a bispecific antibody whichhas specificity for two different epitopes on CD37. The inventors havesurprisingly found that such bispecific antibodies have favorable CDCpotency on CD37 expressing cells compared to treating such cells with acombination of the two monoclonal antibodies which do not compete forbinding to CD37. Additionally, the inventors have surprisingly foundthat such bispecific antibodies have favorable ADCC potency on CD37expressing cells compared to treating such cells with a combination ofthe two monoclonal antibodies which do not compete for binding to CD37.

In one embodiment of the invention the bispecific antibody comprises afirst antigen binding region which is obtained from an antibody whichcompetes for binding to human CD37 with an antibody having the CDRsequences of antibody 010 and a second binding region which is obtainedfrom an antibody which competes for binding to human CD37 with theantibody having the CDR sequences of 016.

In another embodiment of the invention the bispecific antibody comprisesa first antigen binding region which is obtained from an antibody whichcompetes for binding to human CD37 with an antibody having the CDRsequences of antibody 010 and a second binding region which is obtainedfrom an antibody which competes for binding to human CD37 with theantibody having the CDR sequences of 004.

In another embodiment of the invention the bispecific antibody comprisesa first antigen binding region which is obtained from an antibody whichcompetes for binding to human CD37 with an antibody having the CDRsequences of antibody 010 and a second binding region which is obtainedfrom an antibody which competes for binding to human CD37 with theantibody having the CDR sequences of G28.1.

In another embodiment of the invention the bispecific antibody comprisesa first antigen binding region which is obtained from an antibody whichcompetes for binding to human CD37 with an antibody having the CDRsequences of antibody 010 and a second binding region which is obtainedfrom an antibody which competes for binding to human CD37 with theantibody having the CDR sequences of 37.3.

In one embodiment of the invention the bispecific antibody comprises afirst antigen binding region which is obtained from an antibody whichcompetes for binding to human CD37 with an antibody having the CDRsequences of antibody 005 and a second binding region which is obtainedfrom an antibody which competes for binding to human CD37 with theantibody having the CDR sequences of 016.

In another embodiment of the invention the bispecific antibody comprisesa first antigen binding region which is obtained from an antibody whichcompetes for binding to human CD37 with an antibody having the CDRsequences of antibody 005 and a second binding region which is obtainedfrom an antibody which competes for binding to human CD37 with theantibody having the CDR sequences of 004.

In another embodiment of the invention the bispecific antibody comprisesa first antigen binding region which is obtained from an antibody whichcompetes for binding to human CD37 with an antibody having the CDRsequences of antibody 005 and a second binding region which is obtainedfrom an antibody which competes for binding to human CD37 with theantibody having the CDR sequences of G28.1.

In another embodiment of the invention the bispecific antibody comprisesa first antigen binding region which is obtained from an antibody whichcompetes for binding to human CD37 with an antibody having the CDRsequences of antibody 005 and a second binding region which is obtainedfrom an antibody which competes for binding to human CD37 with theantibody having the CDR sequences of 37.3.

Such bispecific antibodies described here may in further embodimentscomprise an Fc-Fc interaction enhancing substitution in both Fc regions(i.e. the Fc regions obtained from the first and second parentalantibody) of the bispecific antibody where the substitution correspondsto E430G in IgG1 when using EU numbering and which substitution enhancesthe Fc-Fc interaction of two or more bispecific antibodies of theinvention upon binding to membrane-bound target. In another embodimentthe Fc-Fc interaction enhancing substitution corresponds to E345K inIgG1 when using EU numbering.

In another embodiment of the invention the bispecific antibody comprisesa second antigen binding region which binds to the same epitope on humanCD37 as a CD37 antibody comprising the CDR sequences selected from thegroup comprising:

-   -   (i) VH CDR1 sequence set forth in SEQ ID NO: 23,        -   VH CDR2 sequence set forth in SEQ ID NO: 24,        -   VH CDR3 sequence set forth in SEQ ID NO: 25,        -   VL CDR1 sequence set forth in SEQ ID NO: 27,        -   VL CDR2 sequence: YAS, and        -   VL CDR3 sequence set forth in SEQ ID NO: 28; [016]    -   (ii) VH CDR1 sequence set forth in SEQ ID NO: 2,        -   VH CDR2 sequence set forth in SEQ ID NO: 3,        -   VH CDR3 sequence set forth in SEQ ID NO: 4,        -   VL CDR1 sequence set forth in SEQ ID NO: 6,        -   VL CDR2 sequence: EAS, and        -   VL CDR3 sequence set forth in SEQ ID NO: 7; [004]    -   (iii) VH CDR1 sequence set forth in SEQ ID NO: 40,        -   VH CDR2 sequence set forth in SEQ ID NO: 41,        -   VH CDR3 sequence set forth in SEQ ID NO: 42,        -   VL CDR1 sequence set forth in SEQ ID NO: 44,        -   VL CDR2 sequence: FAK, and        -   VL CDR3 sequence set forth in SEQ ID NO: 45; [G28.1] and    -   iv) VH CDR1 sequence set forth in SEQ ID NO: 47,        -   VH CDR2 sequence set forth in SEQ ID NO: 48,        -   VH CDR3 sequence set forth in SEQ ID NO: 49,        -   VL CDR1 sequence set forth in SEQ ID NO: 51,        -   VL CDR2 sequence: VAT and        -   VL CDR3 sequence set forth in SEQ ID NO: 52. [37.3]

In another embodiment of the invention the bispecific antibody comprisesa second antigen binding region which binds to the same epitope on humanCD37 as a CD37 antibody comprising the CDR sequences selected from thegroup consisting of:

-   -   a. VH CDR1 sequence set forth in SEQ ID NO: 23,        -   VH CDR2 sequence set forth in SEQ ID NO: 24,        -   VH CDR3 sequence set forth in SEQ ID NO: 25,        -   VL CDR1 sequence set forth in SEQ ID NO: 27,        -   VL CDR2 sequence: YAS, and        -   VL CDR3 sequence set forth in SEQ ID NO: 28; [016]    -   b. VH CDR1 sequence set forth in SEQ ID NO: 2,        -   VH CDR2 sequence set forth in SEQ ID NO: 3,        -   VH CDR3 sequence set forth in SEQ ID NO: 4,        -   VL CDR1 sequence set forth in SEQ ID NO: 6,        -   VL CDR2 sequence: EAS, and        -   VL CDR3 sequence set forth in SEQ ID NO: 7; [004]    -   c. VH CDR1 sequence set forth in SEQ ID NO: 40,        -   VH CDR2 sequence set forth in SEQ ID NO: 41,        -   VH CDR3 sequence set forth in SEQ ID NO: 42,        -   VL CDR1 sequence set forth in SEQ ID NO: 44,        -   VL CDR2 sequence: FAK, and        -   VL CDR3 sequence set forth in SEQ ID NO: 45; [G28.1] and    -   d. VH CDR1 sequence set forth in SEQ ID NO: 47,        -   VH CDR2 sequence set forth in SEQ ID NO: 48,        -   VH CDR3 sequence set forth in SEQ ID NO: 49,        -   VL CDR1 sequence set forth in SEQ ID NO: 51,        -   VL CDR2 sequence: VAT and        -   VL CDR3 sequence set forth in SEQ ID NO: 52. [37.3]

In one embodiment of the invention the second antigen binding region ofthe bispecific antibody has a functional epitope comprising one or moreof the amino acids E124, F162, Q163, V164, L165 and H175 of SEQ ID No:62(CD37).

In one embodiment of the invention said second antigen binding regionbinds to a functional epitope comprising one or more of the amino acidsselected from the group consisting of: E124, F162, Q163, V164, L165 andH175 of SEQ ID No:62 (CD37)

In one embodiment of the invention the second antigen binding region ofthe bispecific antibody binds to a functional epitope on CD37, whereinbinding to a mutant CD37 in which any one or more of the amino acidresidues at positions corresponding to positions E124, F162, Q163, V164,L165 and H175 of SEQ ID No:62 (CD37). has/have been substituted withalanines, is reduced as compared to wild type CD37 having the amino acidsequence set forth in SEQ ID NO: 62; reduced binding being determined aszscore (fold change) in binding of said antibody being lowed that −1.5,wherein zscore (fold change) in binding is calculated as set forth inExample 17.

Accordingly, in one embodiment the invention provides a bispecificantibody comprising a first and a second antigen binding region whereinthe first antigen binding region of the bispecific antibody binds to thesame epitope on human CD37 as an anti-CD37 antibody comprising the CDRsequences of antibody 010 and wherein the second antigen binding regionof the bispecific antibody binds to the same epitope on human CD37 as ananti-CD37 antibody comprising the CDR sequences of antibody 016.

In another embodiment the invention provides a bispecific antibodycomprising a first and a second antigen binding region wherein the firstantigen binding region of the bispecific antibody binds to the sameepitope on human CD37 as an anti-CD37 antibody comprising the CDRsequences of antibody 010 and wherein the second antigen binding regionof the bispecific antibody binds to the same epitope on human CD37 as ananti-CD37 antibody comprising the CDR sequences of antibody 004.

In another embodiment the invention provides a bispecific antibodycomprising a first and a second antigen binding region wherein the firstantigen binding region of the bispecific antibody binds to the sameepitope on human CD37 as an anti-CD37 antibody comprising the CDRsequences of antibody 010 and wherein the second antigen binding regionof the bispecific antibody binds to the same epitope on human CD37 as ananti-CD37 antibody comprising the CDR sequences of antibody G28.1.

In another embodiment the invention provides a bispecific antibodycomprising a first and a second antigen binding region wherein the firstantigen binding region of the bispecific antibody binds to the sameepitope on human CD37 as an anti-CD37 antibody comprising the CDRsequences of antibody 010 and wherein the second antigen binding regionof the bispecific antibody binds to the same epitope on human CD37 as ananti-CD37 antibody comprising the CDR sequences of antibody 37.3.

In another embodiment the invention provides a bispecific antibodycomprising a first and a second antigen binding region wherein the firstantigen binding region of the bispecific antibody binds to the sameepitope on human CD37 as an anti-CD37 antibody comprising the CDRsequences of antibody 005 and wherein the second antigen binding regionof the bispecific antibody binds to the same epitope on human CD37 as ananti-CD37 antibody comprising the CDR sequences of antibody 016.

In yet another embodiment the invention provides a bispecific antibodycomprising a first and a second antigen binding region wherein the firstantigen binding region of the bispecific antibody binds to the sameepitope on human CD37 as an anti-CD37 antibody comprising the CDRsequences of antibody 005 and wherein the second antigen binding regionof the bispecific antibody binds to the same epitope on human CD37 as ananti-CD37 antibody comprising the CDR sequences of antibody 004.

In another embodiment the invention provides a bispecific antibodycomprising a first and a second antigen binding region wherein the firstantigen binding region of the bispecific antibody binds to the sameepitope on human CD37 as an anti-CD37 antibody comprising the CDRsequences of antibody 005 and wherein the second antigen binding regionof the bispecific antibody binds to the same epitope on human CD37 as ananti-CD37 antibody comprising the CDR sequences of antibody G28.1.

In another embodiment the invention provides a bispecific antibodycomprising a first and a second antigen binding region wherein the firstantigen binding region of the bispecific antibody binds to the sameepitope on human CD37 as an anti-CD37 antibody comprising the CDRsequences of antibody 005 and wherein the second antigen binding regionof the bispecific antibody binds to the same epitope on human CD37 as ananti-CD37 antibody comprising the CDR sequences of antibody 37.3.

In a further embodiment of the invention the second antigen bindingregion of the bispecific antibody comprises the CDR sequences selectedfrom the group comprising:

-   -   (i) VH CDR1 sequence set forth in SEQ ID NO: 23,        -   VH CDR2 sequence set forth in SEQ ID NO: 24,        -   VH CDR3 sequence set forth in SEQ ID NO: 25,        -   VL CDR1 sequence set forth in SEQ ID NO: 27,        -   VL CDR2 sequence: YAS, and        -   VL CDR3 sequence set forth in SEQ ID NO: 28; [016]    -   (ii) VH CDR1 sequence set forth in SEQ ID NO: 2,        -   VH CDR2 sequence set forth in SEQ ID NO: 3,        -   VH CDR3 sequence set forth in SEQ ID NO: 4,        -   VL CDR1 sequence set forth in SEQ ID NO: 6,        -   VL CDR2 sequence: EAS, and        -   VL CDR3 sequence set forth in SEQ ID NO: 7; [004]    -   (iii) VH CDR1 sequence set forth in SEQ ID NO: 40,        -   VH CDR2 sequence set forth in SEQ ID NO: 41,        -   VH CDR3 sequence set forth in SEQ ID NO: 42,        -   VL CDR1 sequence set forth in SEQ ID NO: 44,        -   VL CDR2 sequence: FAK, and        -   VL CDR3 sequence set forth in SEQ ID NO: 45; [G28.1] and    -   (iv) VH CDR1 sequence set forth in SEQ ID NO: 47,        -   VH CDR2 sequence set forth in SEQ ID NO: 48,        -   VH CDR3 sequence set forth in SEQ ID NO: 49,        -   VL CDR1 sequence set forth in SEQ ID NO: 51,        -   VL CDR2 sequence: VAT and        -   VL CDR3 sequence set forth in SEQ ID NO: 52. [37.3]

In a further embodiment of the invention the second antigen bindingregion of the bispecific antibody comprises the CDR sequences selectedfrom the group consisting of:

-   -   (i) VH CDR1 sequence set forth in SEQ ID NO: 23,        -   VH CDR2 sequence set forth in SEQ ID NO: 24,        -   VH CDR3 sequence set forth in SEQ ID NO: 25,        -   VL CDR1 sequence set forth in SEQ ID NO: 27,        -   VL CDR2 sequence: YAS, and        -   VL CDR3 sequence set forth in SEQ ID NO: 28; [016]    -   (ii) VH CDR1 sequence set forth in SEQ ID NO: 2,        -   VH CDR2 sequence set forth in SEQ ID NO: 3,        -   VH CDR3 sequence set forth in SEQ ID NO: 4,        -   VL CDR1 sequence set forth in SEQ ID NO: 6,        -   VL CDR2 sequence: EAS, and        -   VL CDR3 sequence set forth in SEQ ID NO: 7; [004]    -   (iii) VH CDR1 sequence set forth in SEQ ID NO: 40,        -   VH CDR2 sequence set forth in SEQ ID NO: 41,        -   VH CDR3 sequence set forth in SEQ ID NO: 42,        -   VL CDR1 sequence set forth in SEQ ID NO: 44,        -   VL CDR2 sequence: FAK, and        -   VL CDR3 sequence set forth in SEQ ID NO: 45; [G28.1] and    -   (iv) VH CDR1 sequence set forth in SEQ ID NO: 47,        -   VH CDR2 sequence set forth in SEQ ID NO: 48,        -   VH CDR3 sequence set forth in SEQ ID NO: 49,        -   VL CDR1 sequence set forth in SEQ ID NO: 51,        -   VL CDR2 sequence: VAT and        -   VL CDR3 sequence set forth in SEQ ID NO: 52. [37.3]

Accordingly, the present invention also provides in another embodiment abispecific antibody comprising a first and a second antigen bindingregion wherein the first antigen binding region of the bispecificantibody comprises the CDR sequences of antibody 010 (i.e. SEQ ID NOs16-18 and 20-21) and wherein the second antigen binding region of thebispecific antibody comprises the CDR sequences of antibody 016 (i.e.SEQ ID NOs 23-25 and 27-28). As also described above, such bispecificantibody of the invention further comprises an Fc-Fc interactionenhancing mutation in the Fc region of the antibody. In one embodimentthis mutation corresponds to a mutation in position E430 or E345 in IgG1when using the EU numbering system. In one embodiment the mutation is anE430G substitution. In another embodiment it is an E345K substitution.

The present invention also provides in another embodiment a bispecificantibody comprising a first and a second antigen binding region whereinthe first antigen binding region of the bispecific antibody comprisesthe CDR sequences of antibody 010 (i.e. SEQ ID NOs 16-18 and 20-21) andwherein the second antigen binding region of the bispecific antibodycomprises the CDR sequences of antibody 004 (i.e. SEQ ID NOs 2-4 and6-7).

The present invention also provides in another embodiment a bispecificantibody comprising a first and a second antigen binding region whereinthe first antigen binding region of the bispecific antibody comprisesthe CDR sequences of antibody 010 (i.e. SEQ ID NOs 16-18 and 20-21) andwherein the second antigen binding region of the bispecific antibodycomprises the CDR sequences of antibody G28.1 (i.e. SEQ ID NOs 40-42 and44-45).

The present invention also provides in another embodiment a bispecificantibody comprising a first and a second antigen binding region whereinthe first antigen binding region of the bispecific antibody comprisesthe CDR sequences of antibody 010 (i.e. SEQ ID NOs 16-18 and 20-21) andwherein the second antigen binding region of the bispecific antibodycomprises the CDR sequences of antibody 37.3 (i.e. SEQ ID NOs 47-49 and51-52).

In a further embodiment of the invention the second antigen bindingregion of the bispecific antibody comprise the VH and VL sequencesselected from the group comprising:

-   -   (i) VH sequence set forth in SEQ ID NO: 22 and VL sequence set        forth in SEQ ID NO: 26 [016] or    -   (ii) VH sequence set forth in SEQ ID NO: 1 and VL sequence set        forth in SEQ ID NO: 5 or    -   (iii) VH sequence set forth in SEQ ID NO: 39 and VL sequence set        forth in SEQ ID NO: 43 [G28.1] or    -   (iv) VH sequence set forth in SEQ ID NO: 46 and VL sequence set        forth in SEQ ID NO: 50 [37.3] or    -   a VH sequence having at least 90% identity, such as at least 95%        identity, such as at least 98% identity, such as at least 99%        identity and a VL sequence having at least 90% identity, such as        at least 95% identity, such as at least 98% identity, such as at        least 99% identity with the VH sequence and VL sequence,        respectively, as set forth in any one of (i) to (iv).

In a further embodiment of the invention the second antigen bindingregion of the bispecific antibody comprise the VH and VL sequencesselected from the group consisting of:

-   -   (i) VH sequence set forth in SEQ ID NO: 22 and VL sequence set        forth in SEQ ID NO: 26 [016],    -   (ii) VH sequence set forth in SEQ ID NO: 1 and VL sequence set        forth in SEQ ID NO: 5 [004],    -   (iii) VH sequence set forth in SEQ ID NO: 39 and VL sequence set        forth in SEQ ID NO: 43 [G28.1],    -   (iv) VH sequence set forth in SEQ ID NO: 46 and VL sequence set        forth in SEQ ID NO: 50 [37.3] and    -   a VH sequence having at least 90% identity, such as at least 95%        identity, such as at least 98% identity, such as at least 99%        identity and a VL sequence having at least 90% identity, such as        at least 95% identity, such as at least 98% identity, such as at        least 99% identity with the VH sequence and VL sequence,        respectively, as set forth in any one of (i) to (iv).

Thus, in another embodiment the present invention also provides abispecific antibody comprising a first and a second antigen bindingregion wherein the first antigen binding region of the bispecificantibody comprises the VH and VL sequences of antibody 010 (i.e. SEQ IDNOs 15 and 19) and wherein the second antigen binding region of thebispecific antibody comprises the VH and VL sequences of antibody 016(i.e. SEQ ID NOs 22 and 26).

In another embodiment the present invention also provides a bispecificantibody comprising a first and a second antigen binding region whereinthe first antigen binding region of the bispecific antibody comprisesthe VH and VL sequences of antibody 010 (i.e. SEQ ID Nos 15 and 19) andwherein the second antigen binding region of the bispecific antibodycomprises the VH and VL sequences of antibody 004 (i.e. SEQ ID NOs 1 and5).

In another embodiment the present invention also provides a bispecificantibody comprising a first and a second antigen binding region whereinthe first antigen binding region of the bispecific antibody comprisesthe VH and VL sequences of antibody 010 (i.e. SEQ ID NOs 15 and 19) andwherein the second antigen binding region of the bispecific antibodycomprises the VH and VL sequences of antibody G28.1 (i.e. SEQ ID NOs 39and 43).

In another embodiment the present invention also provides a bispecificantibody comprising a first and a second antigen binding region whereinthe first antigen binding region of the bispecific antibody comprisesthe VH and VL sequences of antibody 010 (i.e. SEQ ID Nos 15 and 19) andwherein the second antigen binding region of the bispecific antibodycomprises the VH and VL sequences of antibody 37.3 (i.e. SEQ ID Nos 46and 50).

In yet another embodiment the present invention provides a bispecificantibody comprising a first and a second antigen binding region whereinthe first antigen binding region of the bispecific antibody comprisesthe VH and VL sequences of antibody 005 (i.e. SEQ ID NOs 8 and 12) andwherein the second antigen binding region of the bispecific antibodycomprises the VH and VL sequences of antibody 016 (i.e. SEQ ID NOs 22and 26).

In another embodiment the present invention also provides a bispecificantibody comprising a first and a second antigen binding region whereinthe first antigen binding region of the bispecific antibody comprisesthe VH and VL sequences of antibody 005 (i.e. SEQ ID NOs 8 and 12) andwherein the second antigen binding region of the bispecific antibodycomprises the VH and VL sequences of antibody 004 (i.e. SEQ ID NOs 1 and5).

In another embodiment the present invention also provides a bispecificantibody comprising a first and a second antigen binding region whereinthe first antigen binding region of the bispecific antibody comprisesthe VH and VL sequences of antibody 005 (i.e. SEQ ID NOs 8 and 12) andwherein the second antigen binding region of the bispecific antibodycomprises the VH and VL sequences of antibody G28.1 (i.e. SEQ ID NOs 39and 43).

In another embodiment the present invention also provides a bispecificantibody comprising a first and a second antigen binding region whereinthe first antigen binding region of the bispecific antibody comprisesthe VH and VL sequences of antibody 005 (i.e. SEQ ID NOs 8 and 12) andwherein the second antigen binding region of the bispecific antibodycomprises the VH and VL sequences of antibody 37.3 (i.e. SEQ ID NOs 46and 50).

In yet other embodiments of the present invention the VH and VLsequences disclosed above may vary within 90% sequence identity.

In yet a different embodiment the present invention provides a CD37binding molecule comprising one antigen binding region described herein,wherein the CDR sequences are the CDR sequences of one of the antibodies004, 005, 010, 016, 28.1 or 37.3. in one embodiment such molecule onlyhas one antigen binding region. Hereby the binding molecule hasmonovalent binding for CD37. Preferably such molecule comprises anintact Fc region of an immunoglobulin. In one embodiment theCD37-binding molecule comprise a second antigen binding region for anirrelevant target which e.g. may be b12.

Fc-Fc Enhancing Mutations

In one embodiment of the invention the one or more Fc-Fc interactionenhancing mutations in said first and second Fc regions of thebispecific antibody are amino acid substitutions. The Fc region of thebispecific antibody can be said to comprise two different Fc regions,one from each parental anti-CD37 antibody. Alternatively, the bispecificantibody may comprise one or more Fc-Fc interaction enhancing mutationsin each half-molecule. It is to be understood that the Fc-Fc interactionenhancing mutations are symmetrical, i.e., identical mutations are madein the two Fc regions.

In one embodiment the invention provides a bispecific antibody whereinthe one or more Fc-Fc interaction enhancing mutations in said first andsecond Fc regions are amino acid substitutions at one or more positionscorresponding to amino acid positions 430, 440 and 345 in human IgG1when using the EU numbering system. In one embodiment the inventionprovides a bispecific antibody wherein the one or more Fc-Fc interactionenhancing mutations in said first and second Fc regions are amino acidsubstitutions at one or more positions corresponding to amino acidpositions 430, 440 and 345 in human IgG1 when using the EU numberingsystem, with the proviso that the substitution in 440 is 440Y or 440W

In another embodiment the invention provides a bispecific antibodycomprising at least one substitution in said first and second Fc regionsselected from the group comprising: E430G, E345K, E430S, E430F, E430T,E345Q, E345R, E345Y, S440Y and S440W. In a particular preferredembodiment the bispecific antibody comprises at least one substitutionin said first and second Fc regions selected from E430G or E345K,preferably E430G. Hereby bispecific antibodies are provided which willhave enhanced Fc-Fc interaction between different antibodies having saidmutation. It is believed that this mutation cause the antibodies to formoligomers on the target cell and thereby enhancing CDC.

In another embodiment the bispecific antibody contains one furthermutation in said Fc regions which mutation is selected from K439E, S440Kand S440R. A bispecific antibody having an additional mutation of K439Eand a second different antibody or bispecific antibody having anadditional S440K or S440R mutation will form oligomers in alternatingpatterns of the first and the second antibodies. This is thought to bebecause the additional mutations will cause a preference for interactionbetween the first and second antibodies rather than interaction betweenfirst and first or second and second antibodies due to non-covalentbinding between said Fc regions.

It is preferred that the Fc-Fc interaction enhancing mutations in saidfirst and second Fc regions are identical substitutions in said firstand second Fc regions. Accordingly, in one preferred embodiment thebispecific antibodies have the same Fc-Fc interaction enhancing mutationin both Fc regions. The Fc region can also be described as Fc chains sothat an antibody has two Fc chains which make up a common Fc region ofthe antibody. Accordingly, in a preferred embodiment the two Fc chainseach comprise a substitution of a position selected from the group ofpositions corresponding to amino acid positions 430, 440 and 345 inhuman IgG1 when using the EU numbering system. In one embodiment the twoFc chains each comprise an E430G substitution so that a bispecificantibody of the invention comprises two E430G substitutions. In anotherembodiment the two Fc chains each comprise an E345K substitution so thatthe bispecific antibody of the invention comprises two E345Ksubstitutions.

In an embodiment of the invention the bispecific antibody is an IgG1isotype.

In an embodiment of the invention the bispecific antibody is an IgG2isotype.

In an embodiment of the invention the bispecific antibody is an IgG3isotype.

In an embodiment of the invention the bispecific antibody is an IgG4isotype.

In an embodiment of the invention the bispecific antibody is an IgGisotype.

In an embodiment of the invention the bispecific antibody is acombination of the isotypes IgG1, IgG2, IgG3 and IgG4. For example thefirst half antibody obtained from the first parental antibody may be anIgG1 isotype and the second half antibody obtained from the secondparental antibody may be an IgG4 isotype so that the bispecific antibodyis a combination of IgG1 and IgG4. In another embodiment it is acombination of IgG1 and IgG2. In another embodiment it is a combinationof IgG1 and IgG3. In another embodiment it is a combination of IgG2 andIgG3. In another embodiment it is a combination of IgG2 and IgG4. Inanother embodiment it is a combination of IgG3 and IgG4. Typically thecore hinge will be an IgG1 type core hinge having the sequence CPPC butit could be other hinges which are stable and do not allow Fab armexchange in vivo which is the case for the IgG4 core hinge having thesequence CPSC.

In a preferred embodiment the bispecific antibody of the invention is afull length antibody.

In yet another embodiment of the invention the bispecific antibody is ahuman antibody. In yet another embodiment of the invention thebispecific antibody is a humanized antibody. In yet another embodimentof the invention the bispecific antibody is a chimeric antibody. In anembodiment of the invention the bispecific antibody is a combination ofhuman, humanized and chimeric. For example the first half antibodyobtained from the first parental antibody may be a human antibody andthe second half antibody obtained from the second parental antibody maybe a humanized antibody so that the bispecific antibody is a combinationof human and humanized. In a preferred embodiment of the invention thebispecific antibody binds both human and cynomolgus monkey CD37, havingthe sequences set forth in SEQ ID Nos 62 and 63, respectively. This isan advantage as this will allow preclinical toxicology studies to beperformed in the cynomolgus monkey with the same bispecific moleculethat will later be tested in humans. In cases where the antibodiesagainst a human target do not also bind the target in an animal model itis very difficult to perform the preclinical toxicology studies and thenon-clinical safety profile of the molecules, which is a requirement byregulatory authorities.

Bispecific Antibody Formats

The present invention provides bispecific CD37×CD37 antibodies whichefficiently promote CDC- and/or ADCC-mediated killing of CD37-expressingtumor cells such as e.g. B-cell derived tumors. Depending on the desiredfunctional properties for a particular use, particular antigen-bindingregions can be selected from the set of antibodies or antigen-bindingregions provided by the present invention. Many different formats anduses of bispecific antibodies are known in the art, and were reviewed byKontermann; Drug Discov Today, 2015 July; 20(7):838-47 and; MAbs, 2012March-April; 4(2):182-97.

A bispecific antibody according to the present invention is not limitedto any particular bispecific format or method of producing it, however,a bispecific antibody of the invention should have an intact Fc domainin order to induce enhanced Fc-Fc interactions.

Examples of bispecific antibody molecules which may be used in thepresent invention comprise (i) a single antibody that has two armscomprising different antigen-binding regions; (ii) adual-variable-domain antibody (DVD-Ig) where each light chain and heavychain contains two variable domains in tandem through a short peptidelinkage (Wu et al., Generation and Characterization of a Dual VariableDomain Immunoglobulin (DVD-Ig™) Molecule, In: Antibody Engineering,Springer Berlin Heidelberg (2010)); (iii) a so-called “dock and lock”molecule, based on the “dimerization and docking domain” in ProteinKinase A.

In one embodiment, the bispecific antibody of the present invention is across-body or a bispecific antibody obtained via a controlled Fab-armexchange (such as described in WO2011131746 (Genmab)).

Examples of different classes of bispecific antibodies include but arenot limited to (i) IgG-like molecules with complementary CH3 domains toforce heterodimerization; (ii) recombinant IgG-like dual targetingmolecules, wherein the two sides of the molecule each contain the Fabfragment or part of the Fab fragment of at least two differentantibodies; (iii) IgG fusion molecules, wherein full length IgGantibodies are fused to extra Fab fragment or parts of Fab fragment;(iv) Fc fusion molecules, wherein single chain Fv molecules orstabilized diabodies are fused to heavy-chain constant-domains,Fc-regions or parts thereof; (v) Fab fusion molecules, wherein differentFab-fragments are fused together, fused to heavy-chain constant-domains,Fc-regions or parts thereof; and (vi) scFv- and diabody-based and heavychain antibodies (e.g., domain antibodies, nanobodies) wherein differentsingle chain Fv molecules or different diabodies or differentheavy-chain antibodies (e.g. domain antibodies, nanobodies) are fused toan Fc-.

Examples of IgG-like molecules with complementary CH3 domain moleculesinclude but are not limited to the Triomab/Quadroma molecules (TrionPharma/Fresenius Biotech; Roche, WO2011069104), the so-calledKnobs-into-Holes molecules (Genentech, WO9850431), CrossMAbs (Roche,WO2011117329) and the electrostatically-steered molecules (Amgen,EP1870459 and WO2009089004; Chugai, US201000155133; Oncomed,WO2010129304), the LUZ-Y molecules (Genentech, Wranik et al. J. Biol.Chem. 2012, 287(52): 43331-9, doi: 10.1074/jbc.M112.397869. Epub 2012Nov. 1), DIG-body and PIG-body molecules (Pharmabcine, WO2010134666,WO2014081202), the Strand Exchange Engineered Domain body (SEEDbody)molecules (EMD Serono, WO2007110205), the BicIonics molecules (Merus,WO2013157953), FcΔAdp molecules (Regeneron, WO201015792), hingeengineered bispecific IgG1 and IgG2 molecules (Pfizer/Rinat,WO11143545), Azymetric scaffold molecules (Zymeworks/Merck,WO2012058768), mAb-Fv molecules (Xencor, WO2011028952), bivalentbispecific antibodies (WO2009080254) and the DuoBody® molecules (GenmabA/S, WO2011131746).

Examples of recombinant IgG-like dual targeting molecules include butare not limited to Dual Targeting (DT)-Ig molecules (WO2009058383),Two-in-one Antibody (Genentech; Bostrom, et al 2009. Science 323,1610-1614), Cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star,WO2008003116), Zybody molecules (Zyngenia; LaFleur et al. MAbs. 2013March-April; 5(2):208-18), approaches with common light chain(Crucell/Merus, U.S. Pat. No. 7,262,028), κλBodies (NovImmune,WO2012023053) and CovX-body (CovX/Pfizer; Doppalapudi, V. R., et al2007. Bioorg. Med. Chem. Lett. 17, 501-506).

Examples of IgG fusion molecules include but are not limited to DualVariable Domain (DVD)-Ig molecules (Abbott, U.S. Pat. No. 7,612,181),Dual domain double head antibodies (Unilever; Sanofi Aventis,WO20100226923), IgG-like Bispecific molecules (ImClone/Eli Lilly, Lewiset al. Nat Biotechnol. 2014 February; 32(2):191-8), Ts2Ab (MedImmune/AZ;Dimasi et al. J Mol Biol. 2009 Oct. 30; 393(3):672-92) and BsAbmolecules (Zymogenetics, WO2010111625), HERCULES molecules (Biogen Idec,US007951918), scFv fusion molecules (Novartis), scFv fusion molecules(Changzhou Adam Biotech Inc, CN 102250246) and TvAb molecules (Roche,WO2012025525, WO2012025530).

Examples of Fc fusion molecules include but are not limited to ScFv/FcFusions (Pearce et al., Biochem Mol Biol Int. 1997 September;42(6):1179-88), SCORPION molecules (Emergent BioSolutions/Trubion,Blankenship J W, et al. AACR 100 th Annual meeting 2009 (Abstract#5465); Zymogenetics/BMS, WO2010111625), Dual Affinity RetargetingTechnology (Fc-DART) molecules (MacroGenics, WO2008157379, WO2010080538)and Dual(ScFv)2-Fab molecules (National Research Center for AntibodyMedicine—China).

Examples of Fab fusion bispecific antibodies include but are not limitedto F(ab)2 molecules (Medarex/AMGEN; Deo et al J Immunol. 1998 Feb. 15;160(4):1677-86), Dual-Action or Bis-Fab molecules (Genentech, Bostrom,et al 2009. Science 323, 1610-1614), Dock-and-Lock (DNL) molecules(ImmunoMedics, WO2003074569, WO2005004809), Bivalent Bispecificmolecules (Biotecnol, Schoonjans, J Immunol. 2000 Dec. 15;165(12):7050-7) and Fab-Fv molecules (UCB-Celltech, WO 2009040562 A1).

Examples of scFv-, diabody-based and domain antibodies include but arenot limited to Dual Affinity Retargeting Technology (DART) molecules(MacroGenics, WO2008157379, WO2010080538), COMBODY molecules (EpigenBiotech, Zhu et al. Immunol Cell Biol. 2010 August; 88(6):667-75), anddual targeting nanobodies (Ablynx, Hmila et al., FASEB J. 2010).

In one aspect, the bispecific antibody of the invention comprises afirst Fc-region comprising a first CH3 region, and a second Fc-regioncomprising a second CH3 region, wherein the sequences of the first andsecond CH3 regions are different and are such that the heterodimericinteraction between said first and second CH3 regions is stronger thaneach of the homodimeric interactions of said first and second CH3regions. More details on these interactions and how they can be achievedare provided in WO2011131746 and WO2013060867 (Genmab), which are herebyincorporated by reference.

As described further herein, a stable bispecific CD37×CD37 antibody canbe obtained at high yield using a particular method on the basis of onehomodimeric starting CD37 antibody and another homodimeric starting CD37antibody containing only a few, fairly conservative, asymmetricalmutations in the CH3 regions. Asymmetrical mutations mean that thesequences of said first and second CH3 regions contain amino acidsubstitutions at non-identical positions so that the first and secondCH3 regions have different amino acid sequences.

In one aspect, the bispecific antibody as defined in any of theembodiments disclosed herein comprises first and second Fc region,wherein each of said first and second Fc region comprises at least ahinge region, a CH2 and a CH3 region, wherein in said first Fc region atleast one of the amino acids in the positions corresponding to apositions selected from the group consisting of T366, L368, K370, D399,F405, Y407, and K409 in a human IgG1 heavy chain has been substituted,and in said second Fc region at least one of the amino acids in thepositions corresponding to a position selected from the group consistingof T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavychain has been substituted, and wherein said first and said second Fcregions are not substituted in the same positions.

Accordingly, in a preferred embodiment of the invention the first Fcregion of the bispecific antibody comprises a mutation of the amino acidcorresponding to position F405 in human IgG1 and the second Fc region ofthe bispecific antibody comprises a further mutation of the amino acidcorresponding to position K409 in human IgG1. Accordingly, thesemutations are asymmetric compared to the above mentioned Fc-Fcinteraction enhancing mutations.

In one embodiment of the bispecific antibody as defined in any of theembodiments disclosed herein, the first Fc-region has an amino acidsubstitution at position 366, and said second Fc-region has an aminoacid substitution at a position selected from the group consisting of:368, 370, 399, 405, 407 and 409. In one embodiment the amino acid atposition 366 is selected from Ala, Asp, Glu, His, Asn, Val, or Gln.

In one embodiment of the bispecific antibody as defined in any of theembodiments disclosed herein, the first Fc-region has an amino acidsubstitution at position 368, and said second Fc-region has an aminoacid substitution at a position selected from the group consisting of:366, 370, 399, 405, 407 and 409.

In one embodiment of the bispecific antibody as defined in any of theembodiments disclosed herein, the first Fc-region has an amino acidsubstitution at position 370, and said second Fc-region has an aminoacid substitution at a position selected from the group consisting of:366, 368, 399, 405, 407 and 409.

In one embodiment of the bispecific antibody as defined in any of theembodiments disclosed herein, the first Fc-region has an amino acidsubstitution at position 399, and said second Fc-region has an aminoacid substitution at a position selected from the group consisting of:366, 368, 370, 405, 407 and 409.

In one embodiment of the bispecific antibody as defined in any of theembodiments disclosed herein, the first Fc-region has an amino acidsubstitution at position 405, and said second Fc-region has an aminoacid substitution at a position selected from the group consisting of:366, 368, 370, 399, 407 and 409.

In one embodiment of the bispecific antibody as defined in any of theembodiments disclosed herein, the first Fc-region has an amino acidsubstitution at position 407, and said second Fc-region has an aminoacid substitution at a position selected from the group consisting of:366, 368, 370, 399, 405, and 409.

In one embodiment of the bispecific antibody as defined in any of theembodiments disclosed herein, the first Fc-region has an amino acidsubstitution at position 409, and said second Fc-region has an aminoacid substitution at a position selected from the group consisting of:366, 368, 370, 399, 405, and 407.

Accordingly, in one embodiment of the bispecific antibody as defined inany of the embodiments disclosed herein, the sequences of said first andsecond Fc-region contain asymmetrical mutations, i.e. mutations atdifferent positions in the two Fc-regions, e.g. a mutation at position405 in one of the Fc-regions and a mutation at position 409 in the otherFc-region.

In one embodiment of the bispecific antibody as defined in any of theembodiments disclosed herein, the first Fc-region has an amino acidother than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg,His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and saidsecond Fc-region has an amino-acid substitution at a position selectedfrom the group consisting of: 366, 368, 370, 399, 405 and 407. In onesuch embodiment, said first Fc-region has an amino acid other than Lys,Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn,Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said secondFc-region has an amino acid other than Phe, e.g. Gly, Ala, Val, Ile,Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, Cys, Lys, orLeu, at position 405. In a further embodiment hereof, said firstFc-region has an amino acid other than Lys, Leu or Met, e.g. Gly, Ala,Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, orCys, at position 409 and said second Fc-region has an amino acid otherthan Phe, Arg or Gly, e.g. Leu, Ala, Val, Ile, Ser, Thr, Met, Lys, His,Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 405.

In another embodiment of the bispecific antibody as defined in any ofthe embodiments disclosed herein, said first Fc-region comprises a Pheat position 405 and an amino acid other than Lys, Leu or Met, e.g. Gly,Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp,Tyr, or Cys, at position 409 and said second Fc-region comprises anamino acid other than Phe, e.g. Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg,His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, Leu, Met, or Cys, at position405 and a Lys at position 409. In a further embodiment hereof, saidfirst Fc-region comprises a Phe at position 405 and an amino acid otherthan Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His,Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and saidsecond Fc-region comprises an amino acid other than Phe, Arg or Gly,e.g. Leu, Ala, Val, Ile, Ser, Thr, Met, Lys, His, Asp, Asn, Glu, Gln,Pro, Trp, Tyr, or Cys, at position 405 and a Lys at position 409.

In another embodiment of the bispecific antibody as defined in any ofthe embodiments disclosed herein, said first Fc-region comprises a Pheat position 405 and an amino acid other than Lys, Leu or Met, e.g. Gly,Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp,Tyr, or Cys, at position 409 and said second Fc-region comprises a Leuat position 405 and a Lys at position 409. In a further embodimenthereof, said first Fc-region comprises a Phe at position 405 and an Argat position 409 and said second Fc-region comprises an amino acid otherthan Phe, Arg or Gly, e.g. Leu, Ala, Val, Ile, Ser, Thr, Lys, Met, His,Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 405 and a Lys atposition 409. In another embodiment, said first Fc-region comprises Pheat position 405 and an Arg at position 409 and said second Fc-regioncomprises a Leu at position 405 and a Lys at position 409.

In a further embodiment of the bispecific antibody as defined in any ofthe embodiments disclosed herein, said first Fc-region comprises anamino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser,Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, atposition 409 and said second Fc-region comprises a Lys at position 409,a Thr at position 370 and a Leu at position 405. In a furtherembodiment, said first Fc-region comprises an Arg at position 409 andsaid second Fc-region comprises a Lys at position 409, a Thr at position370 and a Leu at position 405.

In an even further embodiment of the bispecific antibody as defined inany of the embodiments disclosed herein, said first Fc-region comprisesa Lys at position 370, a Phe at position 405 and an Arg at position 409and said second Fc-region comprises a Lys at position 409, a Thr atposition 370 and a Leu at position 405.

In another embodiment of the bispecific antibody as defined in any ofthe embodiments disclosed herein, said first Fc-region comprises anamino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser,Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, atposition 409 and said second Fc-region comprises a Lys at position 409and: a) an Ile at position 350 and a Leu at position 405, or b) a Thr atposition 370 and a Leu at position 405.

In another embodiment of the bispecific antibody as defined in any ofthe embodiments disclosed herein, said first Fc-region comprises an Argat position 409 and said second Fc region comprises a Lys at position409 and: a) an Ile at position 350 and a Leu at position 405, or b) aThr at position 370 and a Leu at position 405.

In another embodiment of the bispecific antibody as defined in any ofthe embodiments disclosed herein, said first Fc-region comprises a Thrat position 350, a Lys at position 370, a Phe at position 405 and an Argat position 409 and said second Fc region comprises a Lys at position409 and: a) an Ile at position 350 and a Leu at position 405, or b) aThr at position 370 and a Leu at position 405.

In another embodiment of the bispecific antibody as defined in any ofthe embodiments disclosed herein, said first Fc-region comprises a Thrat position 350, a Lys at position 370, a Phe at position 405 and an Argat position 409 and said second Fc-region comprises an Ile at position350, a Thr at position 370, a Leu at position 405 and a Lys at position409.

In one embodiment of the bispecific antibody as defined in any of theembodiments disclosed herein, said first Fc-region has an amino acidother than Lys, Leu or Met at position 409 and said second Fc-region hasan amino acid other than Phe at position 405, such as other than Phe,Arg or Gly at position 405; or said first CH3 region has an amino acidother than Lys, Leu or Met at position 409 and said second CH3 regionhas an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser orThr at position 407.

In one embodiment, the bispecific antibody as defined in any of theembodiments disclosed herein comprises a first Fc-region having an aminoacid other than Lys, Leu or Met at position 409 and a second Fc-regionhaving an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Seror Thr at position 407.

In one embodiment, the bispecific antibody as defined in any of theembodiments disclosed herein comprises a first Fc-region having a Tyr atposition 407 and an amino acid other than Lys, Leu or Met at position409 and a second Fc-region having an amino acid other than Tyr, Asp,Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407 and a Lys atposition 409.

In one embodiment, the bispecific antibody as defined in any of theembodiments disclosed herein comprises a first Fc-region having a Tyr atposition 407 and an Arg at position 409 and a second Fc-region having anamino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr atposition 407 and a Lys at position 409.

In another embodiment, said first Fc-region has an amino acid other thanLys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp,Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said secondFc-region has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln,Arg, Ser or Thr, e.g. Leu, Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp,or Cys, at position 407. In another embodiment, said first Fc-region hasan amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser,Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, atposition 409 and said second Fc-region has an Ala, Gly, His, Ile, Leu,Met, Asn, Val or Trp at position 407.

In another embodiment of the bispecific antibody as defined in any ofthe embodiments disclosed herein, said first Fc-region has an amino acidother than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg,His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and saidsecond Fc-region has a Gly, Leu, Met, Asn or Trp at position 407.

In another embodiment of the bispecific antibody as defined in any ofthe embodiments disclosed herein, said first Fc-region has a Tyr atposition 407 and an amino acid other than Lys, Leu or Met, e.g. Gly,Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp,Tyr, or Cys, at position 409 and said second Fc-region has an amino acidother than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr, e.g. Leu, Met,Gly, Ala, Val, Ile, His, Asn, Pro, Trp, or Cys, at position 407 and aLys at position 409.

In another embodiment of the bispecific antibody as defined in any ofthe embodiments disclosed herein, said first Fc-region has a Tyr atposition 407 and an amino acid other than Lys, Leu or Met, e.g. Gly,Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp,Tyr, or Cys, at position 409 and said second Fc-region has an Ala, Gly,His, Ile, Leu, Met, Asn, Val or Trp at position 407 and a Lys atposition 409.

In another embodiment of the bispecific antibody as defined in any ofthe embodiments disclosed herein, said first Fc-region has a Tyr atposition 407 and an amino acid other than Lys, Leu or Met, e.g. Gly,Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp,Tyr, or Cys, at position 409 and said second Fc-region has a Gly, Leu,Met, Asn or Trp at position 407 and a Lys at position 409.

In another embodiment of the bispecific antibody as defined in any ofthe embodiments disclosed herein, said first Fc-region has a Tyr atposition 407 and an Arg at position 409 and said second Fc-region has anamino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr,e.g. Leu, Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp, or Cys, atposition 407 and a Lys at position 409.

In another embodiment of the bispecific antibody as defined in any ofthe embodiments disclosed herein, said first Fc-region has a Tyr atposition 407 and an Arg at position 409 and said second Fc-region has anAla, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407 and a Lysat position 409.

In another embodiment of the bispecific antibody as defined in any ofthe embodiments disclosed herein, said first Fc-region has a Tyr atposition 407 and an Arg at position 409 and said second Fc-region has aGly, Leu, Met, Asn or Trp at position 407 and a Lys at position 409.

In another embodiment of the bispecific antibody as defined in any ofthe embodiments disclosed herein, the first Fc-region has an amino acidother than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg,His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409, and thesecond Fc-region has

-   -   (i) an amino acid other than Phe, Leu and Met, e.g. Gly, Ala,        Val, Ile, Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp,        Tyr, or Cys, at position 368, or    -   (ii) a Trp at position 370, or    -   (iii) an amino acid other than Asp, Cys, Pro, Glu or Gln, e.g.        Phe, Leu, Met, Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asn,        Trp, Tyr, or Cys, at position 399 or    -   (iv) an amino acid other than Lys, Arg, Ser, Thr, or Trp, e.g.        Phe, Leu, Met, Ala, Val, Gly, Ile, Asn, His, Asp, Glu, Gln, Pro,        Tyr, or Cys, at position 366.

In one embodiment, the first Fc-region has an Arg, Ala, His or Gly atposition 409, and the second Fc region has

-   -   (i) a Lys, Gln, Ala, Asp, Glu, Gly, His, Ile, Asn, Arg, Ser,        Thr, Val, or Trp at position 368, or    -   (ii) a Trp at position 370, or    -   (iii) an Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, Trp, Phe, His,        Lys, Arg or Tyr at position 399, or    -   (iv) an Ala, Asp, Glu, His, Asn, Val, Gln, Phe, Gly, Ile, Leu,        Met, or Tyr at position 366.

In one embodiment, the first Fc-region has an Arg at position 409, andthe second Fc region has

-   -   (i) an Asp, Glu, Gly, Asn, Arg, Ser, Thr, Val, or Trp at        position 368, or    -   (ii) a Trp at position 370, or    -   (iii) a Phe, His, Lys, Arg or Tyr at position 399, or    -   (iv) an Ala, Asp, Glu, His, Asn, Val, Gln at position 366.

In addition to the above-specified amino-acid substitutions, said firstand second Fc regions may contain further amino-acid substitutions,deletion or insertions relative to wild-type Fc sequences.

In a preferred embodiment of the invention the second Fc region of thebispecific antibody comprises a mutation corresponding to F405 in humanIgG1 and the first Fc region comprises a mutation corresponding to K409in human IgG1 when using EU numbering.

In one embodiment the mutations at position F405 and K409 aresubstitutions. In a particular embodiment the substitution at positionF405 is an F405L substitution. In another embodiment the substitution atposition K409 is a K409R substitution.

In embodiments where the bispecific antibody is an IgG4 isotype thefirst Fc region may further comprise an F405L substitution and an R409Ksubstitution. In such embodiments the second Fc region is notsubstituted in any of 405 and 409 amino acid positions.

It is to be understood that except expressly stated otherwise all thementioned amino acid mutations at the disclosed positions are mutationsrelative to a human IgG1 and using human IgG1 for numbering using the EUnumbering system.

In one embodiment, neither said first nor said second Fc-regioncomprises a Cys-Pro-Ser-Cys sequence in the core hinge region.

In a further embodiment, both said first and said second Fc-regioncomprise a Cys-Pro-Pro-Cys sequence in the core hinge region.

Hereby bispecific antibodies are provided which can be produced in highyields and which are stable in vivo.

In another embodiment the bispecific antibody of the invention hasincreased CDC and/or ADCC effector functions compared to an identicalbispecific antibody which does not have the Fc-Fc interaction enhancingmutations. In another embodiment the bispecific antibody of theinvention has increased CDC and/or ADCC effector functions compared to amonoclonal parental antibody having a binding region of either the firstor the second binding region of the bispecific antibody and havingidentical Fc-Fc enhancing mutations as the bispecific antibody of theinvention.

Method of Preparing Bispecific Antibodies of the Invention

Traditional methods such as the hybrid hybridoma and chemicalconjugation methods (Marvin and Zhu (2005) Acta Pharmacol Sin 26:649)can be used in the preparation of the bispecific antibodies of theinvention. Co-expression in a host cell of two antibodies, consisting ofdifferent heavy and light chains, leads to a mixture of possibleantibody products in addition to the desired bispecific antibody, whichcan then be isolated by, e.g., affinity chromatography or similarmethods.

Strategies favoring the formation of a functional bispecific product,upon co-expression of different antibody constructs can also be used,e.g., the method described by Lindhofer et al. (1995 J Immunol 155:219).Fusion of rat and mouse hybridomas producing different antibodies leadsto a limited number of heterodimeric proteins because of preferentialspecies-restricted heavy/light chain pairing. Another strategy topromote formation of heterodimers over homodimers is a “knob-into-hole”strategy in which a protuberance is introduced on a first heavy-chainpolypeptide and a corresponding cavity in a second heavy-chainpolypeptide, such that the protuberance can be positioned in the cavityat the interface of these two heavy chains so as to promote heterodimerformation and hinder homodimer formation. “Protuberances” areconstructed by replacing small amino-acid side-chains from the interfaceof the first polypeptide with larger side chains. Compensatory“cavities” of identical or similar size to the protuberances are createdin the interface of the second polypeptide by replacing large amino-acidside-chains with smaller ones (U.S. Pat. No. 5,731,168). EP1870459(Chugai) and WO2009089004 (Amgen) describe other strategies for favoringheterodimer formation upon co-expression of different antibody domainsin a host cell. In these methods, one or more residues that make up theCH3-CH3 interface in both CH3 domains are replaced with a charged aminoacid such that homodimer formation is electrostatically unfavorable andheterodimerization is electrostatically favorable. WO2007110205 (Merck)describe yet another strategy, wherein differences between IgA and IgGCH3 domains are exploited to promote heterodimerization.

Another in vitro method for producing bispecific antibodies has beendescribed in WO2008119353 (Genmab), wherein a bispecific antibody isformed by “Fab-arm” or “half-molecule” exchange (swapping of a heavychain and attached light chain) between two monospecific IgG4- orIgG4-like antibodies upon incubation under reducing conditions. Theresulting product is a bispecific antibody having two Fab arms which maycomprise different sequences.

A preferred method for preparing the bispecific CD37×CD37 antibodies ofthe present invention includes the methods described in WO2011131746 andWO2013060867 (Genmab) comprising the following steps:

-   -   a) providing a first antibody comprising an Fc region, said Fc        region comprising a first CH3 region;    -   b) providing a second antibody comprising a second Fc region,        said Fc region comprising a second CH3 region, wherein the first        antibody is a CD37 antibody and the second antibody is a        different CD37 antibody;        wherein the sequences of said first and second CH3 regions are        different and are such that the heterodimeric interaction        between said first and second CH3 regions is stronger than each        of the homodimeric interactions of said first and second CH3        regions;    -   c) incubating said first antibody together with said second        antibody under reducing conditions; and    -   d) obtaining said bispecific antibody.

In one embodiment, said first antibody together with said secondantibody are incubated under reducing conditions sufficient to allow thecysteines in the hinge region to undergo disulfide-bond isomerization,wherein the heterodimeric interaction between said first and secondantibodies in the resulting heterodimeric antibody is such that noFab-arm exchange occurs at 0.5 mM GSH after 24 hours at 37° C.

Without being limited to theory, in step c), the heavy-chain disulfidebonds in the hinge regions of the parent antibodies are reduced and theresulting cysteines are then able to form inter heavy-chain disulfidebond with cysteine residues of another parent antibody molecule(originally with a different specificity). In one embodiment of thismethod, the reducing conditions in step c) comprise the addition of areducing agent, e.g. a reducing agent selected from the group consistingof: 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol(DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine andbeta-mercapto-ethanol, preferably a reducing agent selected from thegroup consisting of: 2-mercaptoethylamine, dithiothreitol andtris(2-carboxyethyl)phosphine. In a further embodiment, step c)comprises restoring the conditions to become non-reducing or lessreducing, for example by removal of a reducing agent, e.g. by desalting.

For this method any of the CD37 antibodies described herein may be usedincluding first and second CD37 antibodies, comprising a first and/orsecond Fc region. Examples of such first and second Fc regions,including combination of such first and second Fc regions may includeany of those described herein.

In one embodiment of this method, said first and/or second antibodiesare full-length antibodies.

The Fc regions of the first and second antibodies may be of any isotype,including, but not limited to, IgG1, IgG2, IgG3 or IgG4. In oneembodiment of this method, the Fc regions of both said first and saidsecond antibodies are of the IgG1 isotype. In another embodiment, one ofthe Fc regions of said antibodies is of the IgG1 isotype and the otherof the IgG4 isotype. In the latter embodiment, the resulting bispecificantibody comprises an Fc region of an IgG1 and an Fc region of IgG4 andmay thus have interesting intermediate properties with respect toactivation of effector functions.

In a further embodiment, one of the antibody starting proteins has beenengineered to not bind Protein A, thus allowing to separate theheterodimeric protein from said homodimeric starting protein by passingthe product over a protein A column.

As described above, the sequences of the first and second CH3 regions ofthe homodimeric starting antibodies (the parental antibodies) aredifferent and are such that the heterodimeric interaction between saidfirst and second CH3 regions is stronger than each of the homodimericinteractions of said first and second CH3 regions. More details on theseinteractions and how they can be achieved are provided in WO2011131746and WO2013060867 (Genmab), which are hereby incorporated by reference intheir entirety.

In particular, a stable bispecific CD37×CD37 antibody can be obtained athigh yield using the above method of the invention on the basis of twohomodimeric starting antibodies which bind different epitopes of CD37and contain only a few, fairly conservative, asymmetrical mutations inthe CH3 regions. Asymmetrical mutations mean that the sequences of saidfirst and second CH3 regions contain amino acid substitutions atnon-identical positions.

The bispecific antibodies of the invention may also be obtained byco-expression of constructs encoding the first and second polypeptidesin a single cell. Thus, in a further aspect, the invention relates to amethod for producing a bispecific antibody, said method comprising thefollowing steps:

-   -   a) providing a first nucleic-acid construct encoding a first        polypeptide comprising a first Fc region and a first        antigen-binding region of a first antibody heavy chain, said        first Fc region comprising a first CH3 region,    -   b) providing a second nucleic-acid construct encoding a second        polypeptide comprising a second Fc region and a second        antigen-binding region of a second antibody heavy chain, said        second Fc region comprising a second CH3 region,        wherein the sequences of said first and second CH3 regions are        different and are such that the heterodimeric interaction        between said first and second CH3 regions is stronger than each        of the homodimeric interactions of said first and second CH3        regions,        optionally wherein said first and second nucleic acid constructs        encode light chain sequences of said first and second antibodies    -   c) co-expressing said first and second nucleic-acid constructs        in a host cell, and    -   d) obtaining said heterodimeric protein from the cell culture.

Thus, the present invention also relates to a recombinant eukaryotic orprokaryotic host cell which produces a bispecific antibody of thepresent invention.

In one embodiment of the present invention, the bispecific antibody isobtained by any of the methods according to the present invention.

Suitable expression vectors, including promoters, enhancers, etc., andsuitable host cells for the production of antibodies are well-known inthe art. Examples of host cells include yeast, bacterial and mammaliancells, such as CHO or HEK cells.

In one embodiment, the bispecific antibody as defined in any of theembodiments disclosed herein comprises a first Fc-region and a secondFc-region, wherein neither said first nor said second Fc-regioncomprises a Cys-Pro-Ser-Cys sequence in the hinge region.

In one embodiment, the bispecific antibody as defined in any of theembodiments disclosed herein comprises a first Fc-region and a secondFc-region, wherein both of said first and said second Fc-region comprisea Cys-Pro-Pro-Cys sequence in the hinge region.

In one embodiment, the bispecific antibody as defined in any of theembodiments disclosed herein comprises a first Fc-region and a secondFc-region, wherein the first and second Fc-regions are human antibodyFc-regions.

In one embodiment, the bispecific antibody as defined in any of theembodiments disclosed herein comprises a first Fc-region and a secondFc-region, wherein the first and second antigen-binding regions comprisehuman antibody VH sequences and, optionally, human antibody VLsequences.

In one embodiment, the bispecific antibody as defined in any of theembodiments disclosed herein comprises a first Fc-region and a secondFc-region, wherein the first and second antigen-binding regions are fromheavy-chain antibodies.

In one embodiment, the bispecific antibody as defined in any of theembodiments disclosed herein comprises a first Fc-region and a secondFc-region, wherein the first and second antigen-binding regions comprisea first and second light chain.

In further embodiments, the co-expression method according to theinvention comprises any of the further features described under the invitro method above.

Parental Antibodies

In another embodiment the invention relates to the parental antibodieswhich are used to prepare the bispecific antibodies of the invention.

Thus, in an embodiment the invention relates to an anti-CD37 antibodybinding to the same epitope on human CD37 as an anti-CD37 antibody whichantibody comprises:

-   -   (i) a VH region comprising a CDR1 sequence set forth in SEQ ID        NO: 16, a CDR2 sequence set forth in SEQ ID NO: 17 and a CDR3        sequence set forth in SEQ ID NO: 18, and a VL region comprising        a CDR1 sequence set forth in SEQ ID NO: 20, and CDR2 sequence:        KAS, and CDR3 sequence set forth in SEQ ID NO: 21[010]; or    -   (ii) a VH region comprising a CDR1 sequence set forth in SEQ ID        NO: 9, a CDR2 sequence set forth in SEQ ID NO:10 and a CDR3        sequence set forth in SEQ ID NO: 11, and a VL region comprising        a CDR1 sequence set forth in SEQ ID NO: 113, and CDR2 sequence:        AAS, and CDR3 sequence set forth in SEQ ID NO: 14[005].

In another embodiment the invention relates to an anti-CD37 antibodywhich competes for binding with an anti-CD37 antibody comprising:

-   -   (i) a VH region comprising a CDR1 sequence set forth in SEQ ID        NO: 16, a CDR2 sequence set forth in SEQ ID NO: 17 and a CDR3        sequence set forth in SEQ ID NO: 18, and a VL region comprising        a CDR1 sequence set forth in SEQ ID NO: 20, and CDR2 sequence:        KAS, and CDR3 sequence set forth in SEQ ID NO: 21 [010]; or    -   (ii) a VH region comprising a CDR1 sequence set forth in SEQ ID        NO: 9, a CDR2 sequence set forth in SEQ ID NO:10 and a CDR3        sequence set forth in SEQ ID NO: 11, and a VL region comprising        a CDR1 sequence set forth in SEQ ID NO: 113, and CDR2 sequence:        AAS, and CDR3 sequence set forth in SEQ ID NO: 14 [005].

In another embodiment the invention relates to an anti-CD37 antibodycomprising:

-   -   (i) a VH region comprising a CDR1 sequence set forth in SEQ ID        NO: 16, a CDR2 sequence set forth in SEQ ID NO: 17 and a CDR3        sequence set forth in SEQ ID NO: 18, and a VL region comprising        a CDR1 sequence set forth in SEQ ID NO: 20, and CDR2 sequence:        KAS, and CDR3 sequence set forth in SEQ ID NO: 21 [010]; or    -   (ii) a VH region comprising a CDR1 sequence set forth in SEQ ID        NO: 9, a CDR2 sequence set forth in SEQ ID NO:10 and a CDR3        sequence set forth in SEQ ID NO: 11, and a VL region comprising        a CDR1 sequence set forth in SEQ ID NO: 113, and CDR2 sequence:        AAS, and CDR3 sequence set forth in SEQ ID NO: 14 [005].

In another embodiment the invention relates to an anti-CD37 antibodywhich comprises:

-   -   (i) a VH region comprising a CDR1 sequence set forth in SEQ ID        NO: 23, a CDR2 sequence set forth in SEQ ID NO: 24 and a CDR3        sequence set forth in SEQ ID NO: 25, and a VL region comprising        a CDR1 sequence set forth in SEQ ID NO: 27, and CDR2 sequence:        YAS, and CDR3 sequence set forth in SEQ ID NO: 28; [016] or    -   (ii) a VH region comprising a CDR1 sequence set forth in SEQ ID        NO: 2, a CDR2 sequence set forth in SEQ ID NO: 3 and a CDR3        sequence set forth in SEQ ID NO: 4, and a VL region comprising a        CDR1 sequence set forth in SEQ ID NO: 6, and CDR2 sequence: EAS,        and CDR3 sequence set forth in SEQ ID NO: 7. [004]

In another embodiment the invention relates to an antibody as definedabove which comprises one or more amino acid mutations in the Fc regionof the antibody which mutation(s) enhances the Fc-Fc interaction betweenthe antibodies upon target binding compared to the Fc-Fc interactionbetween antibodies not having said mutation(s).

It is believed that said enhanced Fc-Fc interaction has the effect thatthe antibodies form oligomers such as e.g. hexamers on the target cellswhich oligomers formation causes the effect of enhanced CDC. In apreferred embodiment the one or more amino acid mutations in the Fcregion of the antibody is amino acid substitutions at one or morepositions corresponding to amino acid positions 430, 440 and 345 inhuman IgG1 when using the EU numbering system and where the substitutionis relative to the amino acid sequence of human IgG1. In one embodimentthe one or more amino acid mutations in the Fc region of the antibody isan amino acid substitution at one or more positions corresponding toamino acid positions 430,345 and 440 in human IgG1 when using the EUnumbering system, with the proviso that the substitution in 440 is 440Yor 440W. In an embodiment the at least one amino acid substitution inthe Fc region is selected from the group comprising: E430G, E345K,E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y and S440W.

In a preferred embodiment the at least one substitution in said Fcregion is selected from E430G or E345K, preferably E430G. These Fc-Fcinteraction enhancing mutations are symmetrical mutations so that thetwo Fc chains of the antibody have identical mutations/substitutions.

In a further embodiment the antibody may further comprise a substitutionat a position corresponding to 366, 368, 370, 399, 405, 407 and 409 inhuman IgG1. An antibody having a substitution in one of these amino acidpositions is a stable antibody; however, it can, under reducingconditions in a so called “fab arm exchange” reaction, form bispecificantibodies with an antibody having a substitution in another of theseamino acid positions and having a different antigen binding region.Under reducing conditions sufficient to allow the cysteines in the hingeregion to undergo disulfide-bond isomerization an antibody of theinvention will form half molecules each comprising a single antigenbinding site and an Fc region. The substitutions at non-identicalpositions in any of positions corresponding to 366, 368, 370, 399, 405,407 and 409 in human IgG1 will cause the half molecules of the firstantibody to favor dimerization with half molecules of the secondantibody so that bispecific (heterodimeric) antibodies will form whenthe reducing conditions are removed and the disulfide bonds in the hingeregion re-forms.

Thus, two antibodies of the present invention having different antigenbinding regions and binding different epitopes on CD37 and containing asubstitution in both Fc chains (Fc regions) in any of the amino acidpositions corresponding to 366, 368, 370, 399, 405, 407 and 409 in humanIgG1 but in non-identical positions may be suitable for preparing abispecific antibody of the invention.

In one embodiment, the first antibody has an amino acid substitution atposition 366, and said second homodimeric protein has an amino acidsubstitution at a position selected from the group consisting of: 368,370, 399, 405, 407 and 409. In one embodiment the amino acid at position366 is selected from Arg, Lys, Asn, Gln, Tyr, Glu and Gly.

In one embodiment, the first antibody has an amino acid substitution atposition 368, and said second antibody has an amino acid substitution ata position selected from the group consisting of: 366, 370, 399, 405,407 and 409.

In one embodiment, the first antibody has an amino acid substitution atposition 370, and said second antibody has an amino acid substitution ata position selected from the group consisting of: 366, 368, 399, 405,407 and 409.

In one embodiment, the first antibody has an amino acid substitution atposition 399, and said second antibody has an amino acid substitution ata position selected from the group consisting of: 366, 368, 370, 405,407 and 409.

In one embodiment, the first antibody has an amino acid substitution atposition 405, and said second antibody has an amino acid substitution ata position selected from the group consisting of: 366, 368, 370, 399,407 and 409.

In one embodiment, the first antibody has an amino acid substitution atposition 407, and said second antibody has an amino acid substitution ata position selected from the group consisting of: 366, 368, 370, 399,405, and 409.

In one embodiment, the first antibody has an amino acid substitution atposition 409, and said second antibody has an amino acid substitution ata position selected from the group consisting of: 366, 368, 370, 399,405, and 407.

In one embodiment, the first antibody has an amino acid other than Lys,Leu or Met at position 409 such as an amino acid selected from the groupcomprising: Gly, Ala, Val, Ile, Ser, Thr, Arg, His, Asp, Asn, Glu, Gln,Trp, Phe, or Tyr at position 409 and said second antibody has anamino-acid substitution at a position selected from the group consistingof: 366, 368, 370, 399, 405 and 407.

In one such embodiment, said first antibody has an amino acid other thanLys, Leu or Met at position 409 such as an amino acid selected from thegroup comprising: Gly, Ala, Val, Ile, Ser, Thr, Arg, His, Asp, Asn, Glu,Gln, Trp, Phe, or Tyr at position 409 and said second antibody has anamino acid other than Phe at position 405 such as an amino acid selectedfrom the group comprising: Gly, Ala, Val, Leu, Ile, Ser, Thr, Lys, Arg,His, Asp, Asn, Glu, Gln, Trp, Met or Tyr at the position correspondingto 405 in IgG1. In a further embodiment hereof, said first antibody hasan amino acid other than Lys, Leu or Met at position 409 and said secondantibody has an amino acid other than Phe, Arg or Gly at position 405.

In another embodiment, said first antibody comprises a Phe at position405 and an amino acid other than Lys, Leu or Met at position 409 andsaid second antibody comprises an amino acid other than Phe at position405 and a Lys at position 409.

In another embodiment, said first antibody comprises Phe at position 405and an Arg at position 409 and said second antibody comprises a Leu atposition 405 and a Lys at position 409. In embodiments where theantibodies are of IgG1, IgG2 or IgG3 isotypes the first antibody maycomprise an F405L substitution and the second antibody may comprise aK409R substitution or vice versa. However, in embodiments where theantibodies are both of the IgG4 isotypes the amino acid in position 409is naturally an Arg (R). Thus, in such embodiments the first antibody isnot substituted in position 409 but naturally has an R409 and the secondantibody comprises F405L and R405K substitutions, or vice versa; thesecond antibody is not substituted in position 409 but naturally has anR409 and the first antibody comprises F405L and R405K substitutions. Inembodiments where one or both of the first and the second antibodies areof the IgG4 isotype

Accordingly, in one embodiment the antibody of the invention maycomprise a substitution corresponding to F405L in human IgG1. In anotherembodiment an antibody of the invention may comprise a substitutioncorresponding to K409R in human IgG1. Such two different antibodies aresuitable for preparing a bispecific antibody of the invention. In aparticularly preferred embodiment a first antibody of the inventioncomprises an F405L and an E430G substitution and a second antibody ofthe invention comprises a K409R and an E430G substitution. Herebyantibodies are provided which may form bispecific antibodies of theinvention which bispecific antibodies comprise a first half moleculecomprising F405L+E430G substitutions and a second half moleculecomprising K409R+E430G when using IgG1 for numbering. In embodimentswhere the isotype is IgG4 the first half molecule comprisesF405L+R409K+E430G substitutions and the second half molecule comprisesan E430G when using IgG4 for numbering. Preferably, in such IgG4embodiments the core hinge region is substituted from a ‘CPSC’ aminoacid sequence to a ‘CPPC’ sequence to make the bispecific antibody morestable in vivo and/or in vitro compared to an IgG4 antibody having theCPSC core hinge.

In one embodiment of the invention the first antibody comprises a VHregion comprising a CDR1 sequence set forth in SEQ ID NO: 16, a CDR2sequence set forth in SEQ ID NO: 17 and a CDR3 sequence set forth in SEQID NO: 18, and a VL region comprising a CDR1 sequence set forth in SEQID NO: 20, and CDR2 sequence: KAS, and CDR3 sequence set forth in SEQ IDNO: 21 [010]; and an Fc region comprising an F405L substitution and onor more Fc-Fc interaction enhancing mutations. In a preferred embodimentthe Fc-Fc interaction enhancing mutations are substitutions at one ormore amino acid positions selected from the group comprising: 430, 345and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y,S440Y and S440W. In a preferred embodiment it is E430G.

In another embodiment of the invention the first antibody comprises a VHregion comprising a CDR1 sequence set forth in SEQ ID NO: 16, a CDR2sequence set forth in SEQ ID NO: 17 and a CDR3 sequence set forth in SEQID NO: 18, and a VL region comprising a CDR1 sequence set forth in SEQID NO: 20, and CDR2 sequence: KAS, and CDR3 sequence set forth in SEQ IDNO: 21 [010]; and an Fc region comprising an K409R substitution and onor more Fc-Fc interaction enhancing mutations. In a preferred embodimentthe Fc-Fc interaction enhancing mutations are substitutions at one ormore amino acid positions selected from the group comprising: 430, 345and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y,S440Y and S440W. In a preferred embodiment it is E430G.

In another embodiment of the invention the first antibody comprises a VHregion comprising a CDR1 sequence set forth in SEQ ID NO: 9, a CDR2sequence set forth in SEQ ID NO:10 and a CDR3 sequence set forth in SEQID NO: 11, and a VL region comprising a CDR1 sequence set forth in SEQID NO: 113, and CDR2 sequence: AAS, and CDR3 sequence set forth in SEQID NO: 14 [005]; and an Fc region comprising an F405L substitution andon or more Fc-Fc interaction enhancing mutations. In a preferredembodiment the Fc-Fc interaction enhancing mutations are substitutionsat one or more amino acid positions selected from the group comprising:430, 345 and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q,E345R, E345Y, S440Y and S440W. In a preferred embodiment it is E430G.

In yet another embodiment of the invention the first antibody comprisesa VH region comprising a CDR1 sequence set forth in SEQ ID NO: 9, a CDR2sequence set forth in SEQ ID NO:10 and a CDR3 sequence set forth in SEQID NO: 11, and a VL region comprising a CDR1 sequence set forth in SEQID NO: 113, and CDR2 sequence: AAS, and CDR3 sequence set forth in SEQID NO: 14 [005]; and an Fc region comprising an K409R substitution andon or more Fc-Fc interaction enhancing mutations. In a preferredembodiment the Fc-Fc interaction enhancing mutations are substitutionsat one or more amino acid positions selected from the group comprising:430, 345 and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q,E345R, E345Y, S440Y and S440W. In a preferred embodiment it is E430G.

In one embodiment of the invention the second antibody comprises a VHregion comprising a CDR1 sequence set forth in SEQ ID NO: 23, a CDR2sequence set forth in SEQ ID NO: 24 and a CDR3 sequence set forth in SEQID NO: 25, and a VL region comprising a CDR1 sequence set forth in SEQID NO: 27, and CDR2 sequence: YAS, and CDR3 sequence set forth in SEQ IDNO: 28 [016]; and an Fc region comprising an F405L substitution and onor more Fc-Fc interaction enhancing mutations. In a preferred embodimentthe Fc-Fc interaction enhancing mutations are substitutions at one ormore amino acid positions selected from the group comprising: 430, 345and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y,S440Y and S440W. In a preferred embodiment it is E430G.

In another embodiment of the invention the second antibody comprises aVH region comprising a CDR1 sequence set forth in SEQ ID NO: 23, a CDR2sequence set forth in SEQ ID NO: 24 and a CDR3 sequence set forth in SEQID NO: 25, and a VL region comprising a CDR1 sequence set forth in SEQID NO: 27, and CDR2 sequence: YAS, and CDR3 sequence set forth in SEQ IDNO: 28 [016]; and an Fc region comprising an K409R substitution and onor more Fc-Fc interaction enhancing mutations. In a preferred embodimentthe Fc-Fc interaction enhancing mutations are substitutions at one ormore amino acid positions selected from the group comprising: 430, 345and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y,S440Y and S440W. In a preferred embodiment it is E430G.

In another embodiment of the invention the second antibody comprises aVH region comprising a CDR1 sequence set forth in SEQ ID NO: 2, a CDR2sequence set forth in SEQ ID NO: 3 and a CDR3 sequence set forth in SEQID NO: 4, and a VL region comprising a CDR1 sequence set forth in SEQ IDNO: 6, and CDR2 sequence: EAS, and CDR3 sequence set forth in SEQ ID NO:7 [004]; and an Fc region comprising an F405L substitution and on ormore Fc-Fc interaction enhancing mutations. In a preferred embodimentthe Fc-Fc interaction enhancing mutations are substitutions at one ormore amino acid positions selected from the group comprising: 430, 345and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y,S440Y and S440W. In a preferred embodiment it is E430G.

In yet another embodiment of the invention the second antibody comprisesa VH region comprising a CDR1 sequence set forth in SEQ ID NO: 2, a CDR2sequence set forth in SEQ ID NO: 3 and a CDR3 sequence set forth in SEQID NO: 4, and a VL region comprising a CDR1 sequence set forth in SEQ IDNO: 6, and CDR2 sequence: EAS, and CDR3 sequence set forth in SEQ ID NO:7 [004]; and an Fc region comprising an K409R substitution and on ormore Fc-Fc interaction enhancing mutations. In a preferred embodimentthe Fc-Fc interaction enhancing mutations are substitutions at one ormore amino acid positions selected from the group comprising: 430, 345and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y,S440Y and S440W. In a preferred embodiment it is E430G.

In another embodiment of the invention the second antibody comprises aVH region comprising a CDR1 sequence set forth in SEQ ID NO: 40, a CDR2sequence set forth in SEQ ID NO: 41 and a CDR3 sequence set forth in SEQID NO: 42, and a VL region comprising a CDR1 sequence set forth in SEQID NO: 44, and CDR2 sequence: FAK, and CDR3 sequence set forth in SEQ IDNO: 45 [G28.1]; and an Fc region comprising an F405L substitution and onor more Fc-Fc interaction enhancing mutations. In a preferred embodimentthe Fc-Fc interaction enhancing mutations are substitutions at one ormore amino acid positions selected from the group comprising: 430, 345and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y,S440Y and S440W. In a preferred embodiment it is E430G.

In yet another embodiment of the invention the second antibody comprisesa VH region comprising a CDR1 sequence set forth in SEQ ID NO: 40, aCDR2 sequence set forth in SEQ ID NO: 41 and a CDR3 sequence set forthin SEQ ID NO: 42, and a VL region comprising a CDR1 sequence set forthin SEQ ID NO: 44, and CDR2 sequence: FAK, and CDR3 sequence set forth inSEQ ID NO: 45 [G28.1]; and an Fc region comprising an K409R substitutionand on or more Fc-Fc interaction enhancing mutations. In a preferredembodiment the Fc-Fc interaction enhancing mutations are substitutionsat one or more amino acid positions selected from the group comprising:430, 345 and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q,E345R, E345Y, S440Y and S440W. In a preferred embodiment it is E430G.

In another embodiment of the invention the second antibody comprises aVH region comprising a CDR1 sequence set forth in SEQ ID NO: 47, a CDR2sequence set forth in SEQ ID NO: 48 and a CDR3 sequence set forth in SEQID NO: 49, and a VL region comprising a CDR1 sequence set forth in SEQID NO: 51, and CDR2 sequence: VAT, and CDR3 sequence set forth in SEQ IDNO: 52 [37.3]; and an Fc region comprising an F405L substitution and onor more Fc-Fc interaction enhancing mutations. In a preferred embodimentthe Fc-Fc interaction enhancing mutations are substitutions at one ormore amino acid positions selected from the group comprising: 430, 345and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y,S440Y and S440W. In a preferred embodiment it is E430G.

In yet another embodiment of the invention the second antibody comprisesa VH region comprising a CDR1 sequence set forth in SEQ ID NO: 47, aCDR2 sequence set forth in SEQ ID NO: 48 and a CDR3 sequence set forthin SEQ ID NO: 49, and a VL region comprising a CDR1 sequence set forthin SEQ ID NO: 51, and CDR2 sequence: VAT, and CDR3 sequence set forth inSEQ ID NO: 52 [37.3]; and an Fc region comprising an K409R substitutionand on or more Fc-Fc interaction enhancing mutations. In a preferredembodiment the Fc-Fc interaction enhancing mutations are substitutionsat one or more amino acid positions selected from the group comprising:430, 345 and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q,E345R, E345Y, S440Y and S440W. In a preferred embodiment it is E430G.

Accordingly, the invention also relates to an anti-CD37 antibody whichbinds to human CD37 which antibody comprises:

-   -   (i) a VH region comprising a CDR1 sequence set forth in SEQ ID        NO: 40, a CDR2 sequence set forth in SEQ ID NO: 41 and a CDR3        sequence set forth in SEQ ID NO: 42, and a VL region comprising        a CDR1 sequence set forth in SEQ ID NO: 44, and CDR2 sequence:        FAK, and CDR3 sequence set forth in SEQ ID NO: 45 [G28.1] or    -   (ii) a VH region comprising a CDR1 sequence set forth in SEQ ID        NO: 47, a CDR2 sequence set forth in SEQ ID NO: 48 and a CDR3        sequence set forth in SEQ ID NO: 49, and a VL region comprising        a CDR1 sequence set forth in SEQ ID NO: 51, and CDR2 sequence:        VAT, and CDR3 sequence set forth in SEQ ID NO: 52 [37.3];    -   (iii) and wherein the antibody of (i) or (ii) comprises an Fc        region comprising at least one amino acid substitution selected        from the group comprising: E430G, E345K, E430S, E430F, E430T,        E345Q, E345R, E345Y, S440Y and S440W; and    -   (iv) wherein optionally the Fc region further comprises a        mutation of either K409R or F405L.

As mentioned above the anti-CD37 antibodies of the invention may be anIgG1, IgG2, IgG3 or IgG4 isotype. In one embodiment the anti-CD37antibody of the invention is of an IgG isotype.

In one embodiment, the antibody of the invention is human, humanized orchimeric.

In one embodiment, the antibody of the invention binds to both the humanand the cynomolgos CD37 antigens.

Further Embodiments of the Invention

In another embodiment the invention relates to a composition comprisinga bispecific antibody of the invention and further comprising amonospecific anti-CD37 antibody, preferably an anti-CD37 antibody havingthe antigen binding region of either the first or second antigen bindingregion of the bispecific antibody.

In one embodiment the invention relates to a pharmaceutical compositioncomprising a bispecific antibody of the invention or an anti-CD37antibody of the invention and a pharmaceutically acceptable carrier.

In another embodiment the invention relates to a bispecific antibody ofthe invention or an antibody of the invention or a composition of theinvention for use as a medicament.

In one embodiment of the invention the bispecific antibody of theinvention is for use in the treatment of cancer, autoimmune disease orinflammatory disorders.

In one embodiment of the invention the anti-CD37 antibody of theinvention is for use in the treatment of cancer, autoimmune disease orinflammatory disorders.

In one embodiment of the invention the composition of the invention isfor use in the treatment of cancer, autoimmune disease or inflammatorydisorders.

In another embodiment the invention relates to a bispecific antibody ofthe invention for use in the treatment of allergy, transplantationrejection or a B-cell malignancy, such as non-Hodgkin lymphoma (NHL),chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantlecell lymphoma (MCL), plasma cell leukemia (PCL), diffuse large B-celllymphoma (DLBCL), or acute lymphoblastic leukemia (ALL).

In another embodiment the invention relates to a bispecific antibody ofthe invention for use in the treatment of rheumatoid arthritis such asacute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis,acute gouty arthritis, acute immunological arthritis, chronicinflammatory arthritis, degenerative arthritis, type II collagen-inducedarthritis, infectious arthritis, Lyme arthritis, proliferativearthritis, psoriatic arthritis, Still's disease, vertebral arthritis,and juvenile-onset rheumatoid arthritis, osteoarthritis, arthritischronica progrediente, arthritis deformans, polyarthritis chronicaprimaria, reactive arthritis, and ankylosing spondylids) systemic lupuserythematosus (SLE) such as cutaneous SLE or subacute cutaneous SLE,neonatal lupus syndrome (NLE), and lupus erythematosis disseminates,multiple sclerosis, inflammatory bowel disease (IBD) which includesulcerative colitis and Crohn's disease, Chronic obstructive pulmonarydisease (COPD), psoriasis, IgA nephropathy, IgM polyneuropathies,myasthenia gravis, diabetes mellitus, Reynaud's syndrome, andglomerulonephritis, pustulosis palmoplantaris (PPP), erosive lichenplanus, pemphigus bullosa, epidermolysis bullosa, contact dermatitis andatopic dermatitis, polyradiculitis including Guillain-Barre syndrome.

In another embodiment the invention relates to an anti-CD37 antibody ofthe invention for use in the treatment of allergy, transplantationrejection or a B-cell malignancy.

In another embodiment the invention relates to an anti-CD37 antibody ofthe invention for use in the treatment of allergy, transplantationrejection or a B-cell malignancy, such as non-Hodgkin lymphoma (NHL),chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantlecell lymphoma (MCL), plasma cell leukemia (PCL), diffuse large B-celllymphoma (DLBCL), or acute lymphoblastic leukemia (ALL).

In another embodiment the invention relates to an anti-CD37 antibody ofthe invention for use in the treatment of rheumatoid arthritis such asacute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis,acute gouty arthritis, acute immunological arthritis, chronicinflammatory arthritis, degenerative arthritis, type II collagen-inducedarthritis, infectious arthritis, Lyme arthritis, proliferativearthritis, psoriatic arthritis, Still's disease, vertebral arthritis,and juvenile-onset rheumatoid arthritis, osteoarthritis, arthritischronica progrediente, arthritis deformans, polyarthritis chronicaprimaria, reactive arthritis, and ankylosing spondylids) systemic lupuserythematosus (SLE) such as cutaneous SLE or subacute cutaneous SLE,neonatal lupus syndrome (NLE), and lupus erythematosis disseminates,multiple sclerosis, inflammatory bowel disease (IBD) which includesulcerative colitis and Crohn's disease, Chronic obstructive pulmonarydisease (COPD), psoriasis, IgA nephropathy, IgM polyneuropathies,myasthenia gravis, diabetes mellitus, Reynaud's syndrome, andglomerulonephritis, pustulosis palmoplantaris (PPP), erosive lichenplanus, pemphigus bullosa, epidermolysis bullosa, contact dermatitis andatopic dermatitis, polyradiculitis including Guillain-Barre syndrome.

In another embodiment the invention relates to the bispecific antibodyof the invention for use in combination with one or more furthertherapeutic agents. In another embodiment of the invention the anti-CD37antibodies of the invention is for use in combination with one or morefurther therapeutic agents. The one or more further therapeutic agentmay e.g. be selected from the group comprising: doxorubicin, cisplatin,bleomycin, carmustine, cyclophosphamide, chlorambucil, bendamustine,vincristine, fludarabine, ibrutinib and an anti-CD 20 antibody such asrituximab, ofatumumab, Obinutuzumab, Veltuzumab, Ocaratuzumab,Ocrelizumab or TRU-015.

In a preferred embodiment of the invention the further therapeutic agentis an anti-CD20 antibody.

In one embodiment of the invention the anti-CD20 antibody is capable ofbinding to human CD20 having the sequences set forth in SEQ ID No: 72.

In one embodiment of the invention the anti-CD20 antibody is capable ofbinding to cynomolgus monkey CD20 having the sequences set forth in SEQID No: 73.

In one embodiment of the invention the anti-CD20 antibody is capable ofbinding to human and cynomolgus monkey CD20 having the sequences setforth in SEQ ID Nos 72 and 73, respectively.

In one embodiment of the invention the anti-CD20 antibody is capable ofbinding to an epitope on human CD20, which does not comprise or requirethe amino acid residues alanine at position 170 or proline at position172, but which comprises or requires the amino acid residues asparagineat position 163 and asparagine at position 166 of SEQ ID No. 72.Examples of such antibodies are the antibodies denoted 2F2 and 7D8 asdisclosed in WO2004035607 (Genmab) and the antibody denoted 2C6 asdisclosed in WO2005103081 (Genmab). The CDR sequences of 7D8 aredisclosed in Table 1.

In one embodiment of the invention the anti-CD20 antibody is capable ofbinding to an epitope on human CD20, which does not comprise or requirethe amino acid residues alanine at position 170 or proline at position172 of SEQ ID No. 72. An example of such an antibody is 11B8 asdisclosed in WO2004035607 (Genmab). The CDR sequences of 11B8 aredisclosed in Table 1.

In one embodiment of the invention the anti-CD20 antibody is capable ofbinding to a discontinuous epitope on human CD20, wherein the epitopecomprises part of the first small extracellular loop and part of thesecond extracellular loop.

In one embodiment of the invention the anti-CD20 antibody is capable ofbinding to a discontinuous epitope on human CD20, wherein the epitopehas residues AGIYAP of the small first extracellular loop and residuesMESLNFIRAHTPY of the second extracellular loop.

Anti-CD20 antibodies may characterize as type-I and type II anti-CD20antibodies. Type I anti-CD20 antibodies, have high CDC and ADCCactivity, but low apoptosis activity, such as ofatumumab (2F2) andrituximab, whereas type II anti-CD20 antibodies, having low or no CDCactivity, but high ADCC and apoptosis activity, such as obinutuzumab and11B8. Also, type I antibodies induce CD20 to redistribute into largedetergent resistant microdomains (rafts), whereas type II antibodies donot.

In one embodiment of the invention the anti-CD20 antibody comprises anantigen-binding region capable of binding to human CD20, wherein theantigen-binding region competes for binding to human CD20 with ananti-CD20 antibody comprising the variable heavy chain (VH) sequence andvariable light chain (VL) as set forth in SEQ ID No 74 and SEQ ID No 78respectively.

In one embodiment of the invention the anti-CD20 antibody comprises anantigen-binding region capable of binding to human CD20, wherein theantigen-binding region competes for binding to human CD20 with ananti-CD20 antibody comprising the variable heavy chain (VH) sequence andvariable light chain (VL) as set forth in SEQ ID No 81 and SEQ ID No 109respectively.

In one embodiment of the invention the anti-CD20 antibody comprises anantigen-binding region capable of binding to human CD20, wherein theantigen-binding region competes for binding to human CD20 with ananti-CD20 antibody comprising the variable heavy chain (VH) sequence andvariable light chain (VL) as set forth in SEQ ID No 94 and SEQ ID No 98respectively.

In one embodiment of the invention the anti-CD20 antibody comprises anantigen-binding region capable of binding to human CD20, wherein theantigen-binding region competes for binding to human CD20 with ananti-CD20 antibody comprising the variable heavy chain (VH) sequence andvariable light chain (VL) as set forth in SEQ ID No 87 and SEQ ID No 91respectively.

In one embodiment of the invention the anti-CD20 antibody comprises anantigen-binding region capable of binding to human CD20, wherein theantigen-binding region competes for binding to human CD20 with ananti-CD20 antibody comprising the variable heavy chain (VH) sequence andvariable light chain (VL) as set forth in SEQ ID No 101 and SEQ ID No105 respectively.

In one embodiment of the invention the anti-CD20 antibody comprises anantigen-binding region capable of binding to human CD20 comprising theCDR sequences:

-   -   VH CDR1 sequence set forth in SEQ ID NO:75,    -   VH CDR2 sequence set forth in SEQ ID NO:76,    -   VH CDR3 sequence set forth in SEQ ID NO:77,    -   VL CDR1 sequence set forth in SEQ ID NO:79    -   VL CDR2 sequence DAS, and    -   VL CDR3 sequence set forth in SEQ ID NO: 80. [7D8]

In one embodiment of the invention the anti-CD20 antibody comprises anantigen-binding region capable of binding to human CD20 comprising theCDR sequences:

-   -   VH CDR1 sequence set forth in SEQ ID NO:82,    -   VH CDR2 sequence set forth in SEQ ID NO:83,    -   VH CDR3 sequence set forth in SEQ ID NO:84,    -   VL CDR1 sequence set forth in SEQ ID NO:85    -   VL CDR2 sequence DAS, and    -   VL CDR3 sequence set forth in SEQ ID NO: 86. [1188]

In one embodiment of the invention the anti-CD20 antibody comprises anantigen-binding region capable of binding to human CD20 comprising theCDR sequences:

-   -   VH CDR1 sequence set forth in SEQ ID NO:95,    -   VH CDR2 sequence set forth in SEQ ID NO:96,    -   VH CDR3 sequence set forth in SEQ ID NO:97,    -   VL CDR1 sequence set forth in SEQ ID NO:99,    -   VL CDR2 sequence ATS, and    -   VL CDR3 sequence set forth in SEQ ID NO: 100. [Rituximab]

In one embodiment of the invention the anti-CD20 antibody comprises anantigen-binding region capable of binding to human CD20 comprising theCDR sequences:

-   -   VH CDR1 sequence set forth in SEQ ID NO:88,    -   VH CDR2 sequence set forth in SEQ ID NO:89,    -   VH CDR3 sequence set forth in SEQ ID NO:90,    -   VL CDR1 sequence set forth in SEQ ID NO:92    -   VL CDR2 sequence DAS, and    -   VL CDR3 sequence set forth in SEQ ID NO: 93. [ofatumumab]

In one embodiment of the invention the anti-CD20 antibody comprises anantigen-binding region capable of binding to human CD20 comprising theCDR sequences:

-   -   VH CDR1 sequence set forth in SEQ ID NO:102,    -   VH CDR2 sequence set forth in SEQ ID NO:103,    -   VH CDR3 sequence set forth in SEQ ID NO:104,    -   VL CDR1 sequence set forth in SEQ ID NO:106    -   VL CDR2 sequence QMS, and    -   VL CDR3 sequence set forth in SEQ ID NO: 107. [obinutuzumab]

In one embodiment of the invention the anti-CD20 antibody comprises anantigen-binding region capable of binding to human CD20 comprising theCDR sequences selected form the group consisting of:

-   -   i) VH CDR1 sequence set forth in SEQ ID NO:75,        -   VH CDR2 sequence set forth in SEQ ID NO:76,        -   VH CDR3 sequence set forth in SEQ ID NO:77,        -   VL CDR1 sequence set forth in SEQ ID NO:79        -   VL CDR2 sequence DAS, and        -   VL CDR3 sequence set forth in SEQ ID NO: 80. [7D8];    -   ii) VH CDR1 sequence set forth in SEQ ID NO:82,        -   VH CDR2 sequence set forth in SEQ ID NO:83,        -   VH CDR3 sequence set forth in SEQ ID NO:84,        -   VL CDR1 sequence set forth in SEQ ID NO:85        -   VL CDR2 sequence DAS, and        -   VL CDR3 sequence set forth in SEQ ID NO: 86. [1188];    -   iii) VH CDR1 sequence set forth in SEQ ID NO:95,        -   VH CDR2 sequence set forth in SEQ ID NO:96,        -   VH CDR3 sequence set forth in SEQ ID NO:97,        -   VL CDR1 sequence set forth in SEQ ID NO:99,        -   VL CDR2 sequence ATS, and        -   VL CDR3 sequence set forth in SEQ ID NO: 100. [Rituximab];    -   iv) VH CDR1 sequence set forth in SEQ ID NO:88,        -   VH CDR2 sequence set forth in SEQ ID NO:89,        -   VH CDR3 sequence set forth in SEQ ID NO:90,        -   VL CDR1 sequence set forth in SEQ ID NO:92        -   VL CDR2 sequence DAS, and        -   VL CDR3 sequence set forth in SEQ ID NO: 93. [ofatumumab];            and    -   v) VH CDR1 sequence set forth in SEQ ID NO:102,        -   VH CDR2 sequence set forth in SEQ ID NO:103,        -   VH CDR3 sequence set forth in SEQ ID NO:104,        -   VL CDR1 sequence set forth in SEQ ID NO:106        -   VL CDR2 sequence QMS, and        -   VL CDR3 sequence set forth in SEQ ID NO: 107.            [obinutuzumab].

In another embodiment the invention relates to use of the bispecificantibody of the invention or the anti-CD37 antibody of the invention forthe manufacture of a medicament. In another embodiment hereof the use isfor the manufacture of a medicament for the treatment of cancer,autoimmune diseases or an inflammatory diseases such as allergy,transplantation rejection or a B-cell malignancy, such as non-Hodgkinlymphoma (NHL), chronic lymphocytic leukemia (CLL), follicular lymphoma(FL), mantle cell lymphoma (MCL), plasma cell leukemia (PCL), diffuselarge B-cell lymphoma (DLBCL), or acute lymphoblastic leukemia (ALL),rheumatoid arthritis such as acute arthritis, chronic rheumatoidarthritis, gout or gouty arthritis, acute gouty arthritis, acuteimmunological arthritis, chronic inflammatory arthritis, degenerativearthritis, type II collagen-induced arthritis, infectious arthritis,Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still'sdisease, vertebral arthritis, and juvenile-onset rheumatoid arthritis,osteoarthritis, arthritis chronica progrediente, arthritis deformans,polyarthritis chronica primaria, reactive arthritis, and ankylosingspondylids) systemic lupus erythematosus (SLE) such as cutaneous SLE orsubacute cutaneous SLE, neonatal lupus syndrome (NLE), and lupuserythematosis disseminates, multiple sclerosis, inflammatory boweldisease (IBD) which includes ulcerative colitis and Crohn's disease,Chronic obstructive pulmonary disease (COPD), psoriasis, IgAnephropathy, IgM polyneuropathies, myasthenia gravis, diabetes mellitus,Reynaud's syndrome, and glomerulonephritis, pustulosis palmoplantaris(PPP), erosive lichen planus, pemphigus bullosa, epidermolysis bullosa,contact dermatitis and atopic dermatitis, polyradiculitis includingGuillain-Barre syndrome.

In another embodiment the invention relates to a method of inducing celldeath, or inhibiting growth and/or proliferation of a tumor cellexpressing CD37 comprising administering to an individual in needthereof an effective amount of a bispecific antibody of the invention oran anti-CD37 antibody of the invention. In certain embodiments themethod is for treating an individual having allergy, transplantationrejection or a B-cell malignancy, such as non-Hodgkin lymphoma (NHL),chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantlecell lymphoma (MCL), plasma cell leukemia (PCL), diffuse large B-celllymphoma (DLBCL), or acute lymphoblastic leukemia (ALL), comprisingadministering to said individual an effective amount of the bispecificantibody of the invention or the anti-CD37 antibody of the invention. Incertain embodiments the method comprises administering one or morefurther therapeutic agents in combination with said antibody or saidbispecific antibody such as e.g. doxorubicin, cisplatin, bleomycin,carmustine, cyclophosphamide, chlorambucil, bendamustine, vincristine,fludarabine, ibrutinib or an anti-CD20 antibody such as rituximab,ofatumumab, obinutuzumab, veltuzumab, ocaratuzumab, ocrelizumab orTRU-015.

In one embodiment of the invention the further therapeutic agent isselected from the group comprising: cyclophosphamide, chlorambucil,bendamustine, ifosfamide, cisplatin, carboplatin, oxaliplatin,carmustine, prednisone, dexamethasone, fludarabine, pentostatin,cladribine, fluorouracil, gemcitabine, cytarabine, methotrexate,pralatrexate, gemcitabine, vincristine, paclitaxel, docetaxel,doxorubicin, mitoxantrone, etoposide, topotecan, irinotecan, bleomycin,CD20-specific rituximab, obinutuzumab and ofatumumab, CD52-specificalemtuzumab, CD30-specific brentuximab, JNJ-63709178, JNJ-64007957,HuMax-IL8, anti-DR5, anti-VEGF, anti-CD38, anti-PD-1, anti-PD-L1,anti-CTLA4, anti-CD40, anti-CD137, anti-GITR, anti-VISTA, antibodiesspecific for other immunomodulatory targets, brentuximab vedotin,HuMax-TAC-ADC, Interferon, thalidomide, lenalidomide, Axicabtageneciloleucel, bortezomib, romidepsin, belinostat, vorinostat, ibrutinib,acalabrutinib, idelalisib, copanlisib, sorafenib, sunitinib, everolimus,recombinant human TRAIL, birinapant, and venetoclax.

In one embodiment of the invention the further therapeutic agent isselected from the group comprising: ibrutinib, rituximab, venetoclax,CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone),bendamustine, fludarabine, cyclophosphamide, and chlorambucil.

In one embodiment of the invention the further therapeutic agent isselected from the group comprising: ibrutinib, rituximab and venetoclax.

In a further aspect, the invention relates to a nucleic acid constructencoding one or more sequences set out in Table 1. In a further aspectthe invention relates to a nucleic acid construct encoding one or moresequences selected from the group comprising SEQ ID NOs: 1, 2, 3, 4, 5,6, 6a, 7, 8, 9, 10, 11, 12, 13, 13a, 14, 15, 16, 17, 18, 19, 20, 20a,21, 22, 23, 24, 25, 26, 27, 27a, 28, 29, 30, 30a and 31.

The invention further relates to a nucleic acid construct encoding theVH and/or VL region of the bispecific antibody or the anti-CD37 antibodyof any of the embodiments herein.

The invention further relates to a nucleic acid construct encoding thebispecific antibody or the anti-CD37 antibody of any of the embodimentsherein.

In a further embodiment the invention relates to an expression vectorcomprising one or more nucleic acid constructs specified above. Inanother embodiment the invention relates to a host cell comprising anexpression vector as defined above.

An expression vector in the context of the present invention may be anysuitable vector, including chromosomal, non-chromosomal, and syntheticnucleic acid vectors (a nucleic acid sequence comprising a suitable setof expression control elements). Examples of such vectors includederivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeastplasmids, vectors derived from combinations of plasmids and phage DNA,and viral nucleic acid (RNA or DNA) vectors. In one embodiment, a CD37antibody-encoding nucleic acid is comprised in a naked DNA or RNAvector, including, for example, a linear expression element (asdescribed in for instance Sykes and Johnston, Nat Biotech 17, 35559(1997)), a compacted nucleic acid vector (as described in for instanceU.S. Pat. No. 6,077,835 and/or WO 00/70087), a plasmid vector such aspBR322, pUC 19/18, or pUC 118/119, a “midge” minimally-sized nucleicacid vector (as described in for instance Schakowski et al., Mol Ther 3,793800 (2001)), or as a precipitated nucleic acid vector construct, suchas a CaP04-precipitated construct (as described in for instanceWO200046147, Benvenisty and Reshef, PNAS USA 83, 955155 (1986), Wigleret al., Cell 14, 725 (1978), and Coraro and Pearson, Somatic CellGenetics 7, 603 (1981)). Such nucleic acid vectors and the usage thereofare well known in the art (see for instance U.S. Pat. Nos. 5,589,466 and5,973,972).

In one embodiment, the vector is suitable for expression of the CD37antibodies in a bacterial cell. Examples of such vectors includeexpression vectors such as BlueScript (Stratagene), pIN vectors (VanHeeke & Schuster, J Biol Chem 264, 55035509 (1989), pET vectors(Novagen, Madison Wis.) and the like).

An expression vector may also or alternatively be a vector suitable forexpression in a yeast system. Any vector suitable for expression in ayeast system may be employed. Suitable vectors include, for example,vectors comprising constitutive or inducible promoters such as alphafactor, alcohol oxidase and PGH (reviewed in: F. Ausubel et al., ed.Current Protocols in Molecular Biology, Greene Publishing and WileyInterScience New York (1987), and Grant et al., Methods in Enzymol 153,516544 (1987)).

An expression vector may also or alternatively be a vector suitable forexpression in mammalian cells, e.g. a vector comprising glutaminesynthetase as a selectable marker, such as the vectors described inBebbington (1992) Biotechnology (NY) 10:169-175.

A nucleic acid and/or vector may also comprises a nucleic acid sequenceencoding a secretion/localization sequence, which can target apolypeptide, such as a nascent polypeptide chain, to the periplasmicspace or into cell culture media. Such sequences are known in the art,and include secretion leader or signal peptides.

The expression vector may comprise or be associated with any suitablepromoter, enhancer, and other expression-facilitating elements. Examplesof such elements include strong expression promoters (e. g., human CMVIE promoter/enhancer as well as RSV, SV40, SL33, MMTV, and HIV LTRpromoters), effective poly (A) termination sequences, an origin ofreplication for plasmid product in E. coli, an antibiotic resistancegene as selectable marker, and/or a convenient cloning site (e.g., apolylinker). Nucleic acids may also comprise an inducible promoter asopposed to a constitutive promoter such as CMV IE.

In one embodiment, the CD37 antibody-encoding expression vector may bepositioned in and/or delivered to the host cell or host animal via aviral vector.

Thus the present invention also relates to a recombinant eukaryotic orprokaryotic host cell which produces a bispecific antibody of thepresent invention, such as a transfectoma.

The invention further relates to an anti-idiotypic antibody, which bindsto the antigen binding region of the antibody or the bispecific antibodyof the invention.

An in vitro method for detecting the presence of a human CD37 antigen ora cell expressing human CD37 in a sample, said method comprising:

-   -   (i) contacting the sample with the bispecific antibody of any of        the above embodiments or the antibody of any of the embodiments        herein under conditions that allow for formation of a complex        between the antibody or the bispecific antibody and CD37; and    -   (ii) detecting the formation of a complex.

An in vivo method for detecting the presence of a human CD37 antigen, ora cell expressing human CD37 in a subject, said method comprising:

-   -   (i) administering the bispecific antibody of any of the above        embodiments or the antibody of any of the embodiments herein        under conditions that allow for formation of a complex between        the antibody or the bispecific antibody and CD37; and    -   (ii) detecting the formed complex.        Sequences

TABLE 1 SEQ ID NO: LABEL SEQUENCE   1 VH-004-H5L2EVQLVESGGGLVQPGGSLRLSCAASGFSLSTYDMSWVRQAPGKGLEWVSIIYSSVGAYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREYGASSSDYIFSLWGQGTLVTVSS   2 VH-004-H5L2- GFSLSTYD CDR1   3VH-004-H5L2- IYSSVGA CDR2   4 VH-004-H5L2- AREYGASSSDYIFSL CDR3   5VL-004-H5L2 AQVLTQSPSPLSASVGDRVTITCQASQSVYNSQNLAWYQQKPGKAPKLLIYEASKLASGVPSRFKGSGSGTEFTLTISSLQPDDFATYYCQGEFS CISADCTAFGGGTKVEIK   6VH-004-H5L2- QSVYNSQN CDR1 VH-004-H5L2- EAS CDR2   7 VH-004-H5L2-QGEFSCISADCTA CDR3   8 VH-005-H1L2EQSVVESGGGLVQPGGSLRLSCTVSGFSLSSNAMNWVRQAPGKGLEWIGLIYASGNTDYASWAKGRFTISKTSTTVYLKITSPTAEDTATYFCA REGSVWGAAFDPWGQGTLVTVSS  9 VH-005-H1L2- GFSLSSNA CDR1  10 VH-005-H1L2- IYASGNT CDR2  11VH-005-H1L2- AREGSVWGAAFDP CDR3  12 VL-005-H1L2AYDMTQSPSSVSASVGDRVTITCQASQSISNWLAWYQQKPGKAPKQLIYAASTLASGVPSRFKGSGSGTDFTLTISSLQPEDFATYYCQQGYS NSNIDNTFGGGTKVEIK  13VL-005-H1L2- QSISNW CDR1 VL-005-H1L2- AAS CDR2  14 VL-005-H1L2-QQGYSNSNIDNT CDR3  15 VH-010-H5L2EVQLVESGGGLVQPGGSLRLSCAASGFSLSYNAMNWVRQAPGKGLEWVSIIFASGRTDYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGSTWGDALDPWGQGTLVTVSS  16 VH-010-H5L2- GFSLSYNA CDR1  17VH-010-H5L2- IFASGRT CDR2  18 VH-010-H5L2- AREGSTWGDALDP CDR3  19VL-010-H5L2 AYDMTQSPSTLSASVGDRVTITCQASQNIIDYLAWYQQKPGKAPKLLIHKASTLASGVPSRFKGSGSGTEFTLTISSLQPDDFATYYCQQGYSNS NIDNTFGGGTKVEIK  20VL-010-H5L2- QNIIDY CDR1 VL-010-H5L2- KAS CDR2  21 VL-010-H5L2-QQGYSNSNIDNT CDR3  22 VH-016-H5L2EVQLVESGGGLVQPGGSLRLSCAASGFSLSNYNMGWVRQAPGKGLEWVSVIDASGTTYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCARELLYFGSSYYDLWGQGTLVTVSS  23 VH-016-H5L2- GFSLSNYN CDR1  24VH-016-H5L2- IDASGTT CDR2  25 VH-016-H5L2- ARELLYFGSSYYDL CDR3  26VL-016-H5L2 DVVMTQSPSTLSASVGDRVTITCQASQNIDSNLAWYQQKPGKAPKFLIYYASNLPFGVPSRFKGSGSGTEFTLTISSLQPDDFATYYCQCADVG STYVAAFGGGTKVEIK  27VL-016-H5L2- QNIDSN CDR1 VL-016-H5L2- YAS CDR2  28 VL-016-H5L2-QCADVGSTYVAA CDR3  29 VL-016-H5L2-DVVMTQSPSTLSASVGDRVTITCQASQNIDSNLAWYQQKPGKAPKF C90SLIYYASNLPFGVPSRFKGSGSGTEFTLTISSLQPDDFATYYCQ S ADVG STYVAAFGGGTKVEIK  30VL-016-H5L2- QNIDSN C90S-CDR1 VL-016-H5L2- YAS C90S-CDR2  31VL-016-H5L2- QSADVGSTYVAA C90S-CDR3  32 VH-b12QVQLVQSGAEVKKPGASVKVSCQASGYRFSNFVIHWVRQAPGQRFEWMGWINPYNGNKEFSAKFQDRVTFTADTSANTAYMELRSLRSADTAVYYCARVGPYSWDDSPQDNYYMDVWGKGTTVIVSS  33 VH-b12-CDR1 GYRFSNFV  34VH-b12-CDR2 INPYNGNK  35 VH-b12-CDR3 ARVGPYSWDDSPQDNYYMDV  36 VL-b12EIVLTQSPGTLSLSPGERATFSCRSSHSIRSRRVAWYQHKPGQAPRLVIHGVSNRASGISDRFSGSGSGTDFTLTITRVEPEDFALYYCQVYGAS SYTFGQGTKLERK  37VL-b12-CDR1 HSIRSRR VL-b12-CDR2 GVS  38 VL-b12-CDR3 QVYGASSYT  39VH-G28.1 AVQLQQSGPELEKPGASVKISCKASGYSFTGYNMNWVKQNNGKSLEWIGNIDPYYGGTTYNRKFKGKATLTVDKSSSTAYMQLKSLTSEDSAV YYCARSVGPMDYWGQGTSVTVSS 40 VH-G28.1-CDR1 GYSFTGYN  41 VH-G28.1-CDR2 IDPYYGGT  42 VH-G28.1-CDR3ARSVGPMDY  43 VL-G28.1 DIQMTQSPASLSASVGETVTITCRTSENVYSYLAWYQQKQGKSPQLLVSFAKTLAEGVPSRFSGSGSGTQFSLKISSLQPEDSGSYFCQHHSDN PWTFGGGTELEIK  44VL-G28.1-CDR1 ENVYSY VL-G28.1-CDR2 FAK  45 VL-G28.1-CDR3 QHHSDNPWT  46VH-37.3 QVQVKESGPGLVAPSQSLSITCTVSGFSLTTSGVSWVRQPPGKGLEWLGVIWGDGSTNYHSALKSRLSIKKDHSKSQVFLKLNSLQTDDTAT YYCAKGGYSLAHWGQGTLVTVSA 47 VH-37.3-CDR1 GFSLTTSG  48 VH-37.3-CDR2 IWGDGST  49 VH-37.3-CDR3AKGGYSLAH  50 VL-37.3 DIQMTQSPASLSVSVGETVTITCRASENIRSNLAWYQQKQGKSPQLLVNVATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHYWG TTWTFGGGTKLEIK  51VL-37.3-CDR1 ENIRSN VL-37.3-CDR2 VAT  52 VL-37.3-CDR3 QHYWGTTWT  53IgG1-Fc ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK  54 IgG1-Fc-delKASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.  55 IgG1-E430G-FcASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH G ALHNHYTQKSLSLSPGK  56 IgG1-E345R-FcASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR R PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K  57 IgG1-F405L-FcASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSF LLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK  58 IgG1-K409R-FcASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYS RLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK  59 IgG1-F405L-ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT E430G-FcSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSF LLYSKLTVDKSRWQQGNVFSCSVMH G ALHNHYTQKSLSLSPGK  60 IgG1-K409R-ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT E430G-FcSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYS RLTVDKSRWQQGNVFSCSVMH G ALHNHYTQKSLSLSPGK  61 Kappa-CRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC  62Human CD37 MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKVLAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRAQLERSLRDVVEKTIQKYGTNPEETAAEESWDYVQFQLRCCGWHYPQDWFQVLILRGNGSEAHRVPCSCYNLSATNDSTILDKVILPQLSRLGHLARSRHSADICAVPAESHIYREGCAQGLQKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARYR  63 CynomolgusMSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLA CD37 (mfCD37)FVPLQIWSKVLAISGVFTMGLALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRAQLERSLQDIVEKTIQKYHTNPEETAAEESWDYVQFQLRCCGWHSPQDWFQVLTLRGNGSEAHRVPCSCYNLSATNDSTILDKVILPQLSRLGQLARSRHSTDICAVPANSHIYREGCARSLQKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARYR  64 CD37EC2-FcHisMWWRLWWLLLLLLLLWPMVWARAQLERSLRDVVEKTIQKYGTNPEETAAEESWDYVQFQLRCCGWHYPQDWFQVLILRGNGSEAHRVPCSCYNLSATNDSTILDKVILPQLSRLGHLARSRHSADICAVPAESHIYREGCAQGLQKWLHNNPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTAPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGKHHHHHHHH  65CD37MfEC2-  MWWRLWWLLLLLLLLWPMVWARAQLERSLQDIVEKTIQKYHTNPEE FcHisTAAEESWDYVQFQLRCCGWHSPQDWFQVLTLRGNGSEAHRVPCSCYNLSATNDSTILDKVILPQLSRLGQLARSRHSTDICAVPANSHIYREGCARSLQKWLHNNPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTAPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGKHHHHHHHH  66IgG1-F405L- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT E345RSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRRPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K  67 IgG1-F405L-ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT E345KSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRKPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK  68 IgG1-F405L-ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT E430SSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHSALHNHYTQKSLSLSPGK  69 IgG1-K409R-ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT E345RSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRRPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K  70 IgG1-K409R-ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT E345KSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRKPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK  71 IgG1-K409R-ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT E430SSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHSALHNHYTQKSLSLSPGK  72 Human CD20MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLGAVQIMNGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYIISGSLLAATEKNSRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQSLFLGILSVMLIFAFFQELVIAGIVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIEN DSSP  73 CynomolgusMTTPRNSVNGTFPAEPMKGPIAMQPGPKPLLRRMSSLVGPTQSFFMR monkey CD20ESKALGAVQIMNGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYIISGSLLAATEKNSRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKISHFLKMESLNFIRVHTPYINIYNCEPANPSEKNSPSTQYCYSIQSLFLGILSVMLIFAFFQELVIAGIVENEWRRTCSRPKSSVVLLSAEEKKEQVIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIEN DSSP  74 VH CD20-7D8EVQLVESGGGLVQPDRSLRLSCAAS GFTFHDYA MHWVRQAPGKGL EWVST ISWNSGTIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDT ALYYC AKDIQYGNYYYGMDV WGQGTTVTVSS  75VH CD20-7D8 GFTFHDYA CDR1  76 VH CD20-7D8 ISWNSGTI CDR2  77 VH CD20-7D8AKDIQYGNYYYGMDV CDR3  78 VL CD20-7D8 EIVLTQSPATLSLSPGERATLSCRAS QSVSSYLAWYQQKPGQAPRLL IY DAS NRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNW PITFGQGTRLEIK  79 VL CD20-7D8 QSVSSY CDR1 VL CD20-7D8 DAS CDR2  80VL CD20-7D8 QQRSNWPIT CDR3  81 VH CD20-11B8 EVQLVQSGGGLVHPGGSLRLSCTGSGFTFSYHA MHWVRQAPGKGL EWVSI IGTGGVT YYADSVKGRFTISRDNVKNSLYLQMNSLRAEDMAVYYC ARDYYGAGSFYDGLYGMDV WGQGTTVTVSS  82 VH CD20-11B8 GFTFSYHA CDR1  83VH CD20-11B8 IGTGGVT CDR2  84 VH CD20-11B8 ARDYYGAGSFYDGLYGMDV CDR3  85VL CD20-11B8 QSVSSY CDR1 VL CD20-11B8 DAS CDR2  86 VL CD20-11B8QQRSDWPLT CDR3  87 VH CD20- EVQLVESGGGLVQPGRSLRLSCAAS GFTFNDYAMHWVRQAPGKGL ofatumumab EWVST ISWNSGSI GYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYC AKDIQYGNYYYGMDV WGQGTTVTVSS  88 VL CD20- GFTFNDYA ofatumumab CDR1 89 VH CD20- ISWNSGSI ofatumumab CDR2  90 VH CD20- AKDIQYGNYYYGMDVofatumumab CDR3  91 VL CD20- EIVLTQSPATLSLSPGERATLSCRAS QSVSSYLAWYQQKPGQAPRLL ofatumumab IY DAS NRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNW PIT FGQGTRLEIK  92 VL CD20- QSVSSY ofatumumab CDR1 VL CD20- DASofatumumab  93 VL CD20- QQRSNWPIT ofatumumab CDR3  94 VH CD20-QVQLQQPGAELVKPGASVKMSCKAS GYTFTSYN MHWVKQTPGRG rituximab LEWIGA IYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSED SAVYYC ARST YYGGDWYFNV WGAGTTVTVSA  95VH CD20- GYTFTSYN rituximab CDR1  96 VH CD20- IYPGNGDT rituximab CDR2 97 VH CD20- ARSTYYGGDWYFNV rituximab CDR3  98 VL CD20-QIVLSQSPAILSASPGEKVTMTCRAS SSVSY IHWFQQKPGSSPKPWI rituximab Y ATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYC QQWTSN PPT FGGGTKLEIK  99 VL CD20-SSVSY rituximab CDR1 VL CD20- ATS rituximab CDR2 100 VL CD20- QQWTSNPPTrituximab CDR3 101 VH CD20- QVQLVQSGAEVKKPGSSVKVSCKAS GYAFSYSWINWVRQAPGQG obinutuzumab LEWMGR IFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSED TAVYYC ARNVFDGYWLVY WGQGTLVTVSS 102VH CD20- GYAFSYSW obinutuzumab CDR1 103 VH CD20- IFPGDGDT obinutuzumabCDR2 104 VH CD20- ARNVFDGYWLVY obinutuzumab CDR3 105 VL CD20-DIVMTQTPLSLPVTPGEPASISCRSS KSLLHSNGITY LYWYLQKPGQ obinutuzumab SPQLLIYQMS NLVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC A QNLELPYT FGGGTKVEIK 106VL CD20- KSLLHSNGITY obinutuzumab CDR1 VL CD20- QMS obinutuzumab CDR2107 VL CD20- AQNLELPYT obinutuzumab CDR3 108 IgG1-ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS S239D-I332EGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 109 VL CD20 11B8EIVLTQSPATLSLSPGERATLSCRAS QSVSSY LAWYQQKPGQAPRLLIY DAS NRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSDWPLT FGGGTKVEIK 110 VH CD37-004QSVEESGGRLVTPGTPLTLTCTVSGFSLSTYDMSWVRQAPGKGLEWIGIIYSSVGAYYASWAKGRFTFSKTSTTVDLKITSPTTEDTATYFCAREY GASSSDYIFSLWGQGTLVTVSS  2 VH CD37-004 GFSLSTYD CDR1   3 VH CD37-004 IYSSVGA CDR2   4VH CD37-004 AREYGASSSDYIFSL CDR3 111 VL CD37-004AQVLTQTPSPVSAAVGGTVTINCQASQSVYNSQNLAWYQQKPGQPPKLLIYEASKLASGVPSRFKGSGSGTQFTLTISGVQSDDAATYYCQGEF SCISADCTAFGGGTEVVVK   6VL CD37-004 QSVYNSQN CDR1 VL CD37-004 EAS CDR2   7 VL CD37-004QGEFSCISADCTA CDR3 112 VH CD37-005QSVEESGGRLVTPGTPLTLTCTVSGFSLSSNAMNWVRQAPGKGLEWIGLIYASGNTDYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARE GSVWGAAFDPWGPGTLVTVSS  9 VH CD37-005 GFSLSSNA CDR1  10 VH CD37-005 IYASGNT CDR2  11VH CD37-005 AREGSVWGAAFDP CDR3 113 VL CD37-005AYDMTQTPASVEVAVGGTVTIKCQASQSISNWLAWYQQKPGQPPKQLIYAASTLASGVPSRFKGSGSGTQFTLTISGVESADAATYYCQQGYSN SNIDNTFGGGTEVVVK  13VL CD37-005 QSISNW CDR1 VL CD37-005 AAS CDR2  14 VL CD37-005QQGYSNSNIDNT CDR3 114 VH CD37-010QSVEESGGRLVTPGTPLTLTCTVSGFSLSYNAMNWVRQAPGKGLEWIGIIFASGRTDYASWAKGRFTISKTSTTVELKITSPTTEDTATYFCAREG STWGDALDPWGPGTLVTVSS 16 VH CD37-010 GFSLSYNA CDR1  17 VH CD37-010 IFASGRT CDR2  18VH CD37-010 AREGSTWGDALDP CDR3 115 VL CD37-010AYDMTQTPSSVEAAVGGTVTIKCQASQNIIDYLAWYQQKPGQPPQLLIHKASTLASGVPSRFKGSGSGTQFTLTISGVQSDDAATYYCQQGYSN SNIDNTFGGGTEVVVK  20VL CD37-010 QNIIDY CDR1 VL CD37-010 KAS CDR2  21 VL CD37-010QQGYSNSNIDNT CDR3 116 VH CD37-016QSVEESGGRLVTPGTPLTLTCTVSGFSLSNYNMGWVRQAPGKGLEWIGVIDASGTTYYATWAKGRFTCSKTSSTVELKMTSLTTEDTATYFCAR ELLYFGSSYYDLWGQGTLVTVSS 23 VH CD37-016 GFSLSNYN CDR1  24 VH CD37-016 IDASGTT CDR2  25VH CD37-016 ARELLYFGSSYYDL CDR3 117 VL CD37-016DVVMTQTPASVSEPVGGTVTIKCQASQNIDSNLAWYQQKPGQPPKFLIYYASNLPFGVSSRFKGSGSGTQFTLTISDLESADAATYYCQCADVG STYVAAFGGGTEVVVK  27VL CD37-016 QNIDSN CDR1 VL CD37-016 YAS CDR2  28 VL CD37-016QCADVGSTYVAA CDR3

EXAMPLES Example 1: Generation of CD37 Specific Antibodies in Rabbits

Expression Constructs for CD37

The following codon-optimized constructs for expression of full-lengthCD37 variants were generated: human (Homo sapiens) CD37 (Genbankaccession no. NP_001765) (SEQ ID NO: 62), cynomolgus monkey (Macacafascicularis) CD37 ((mfCD37) (SEQ ID NO: 63). In addition, the followingcodon-optimized constructs for expression of various CD37 ECD variantswere generated: a signal peptide encoding sequence followed by thesecond extracellular domain (EC2) of human CD37 (aa 112-241), fused tothe Fc (CH2-CH3) domain of human IgG with a C-terminal His tag(CD37EC2-FcHis, SEQ ID NO: 64), and a similar construct for mfCD37(CD37mfEC2-FcHis, SEQ ID NO: 65). The constructs contained suitablerestriction sites for cloning and an optimal Kozak (GCCGCCACC) sequence[Kozak et al. (1999) Gene 234: 187-208]. The constructs were cloned inthe mammalian expression vector pcDNA3.3 (Invitrogen) or an equivalentvector.

Transient Expression in CHO and HEK Cells

Membrane proteins were transiently transfected in Freestyle 293-F(HEK293F) cells (Life technologies, USA) using 293fectin (Lifetechnologies) essentially as described by the manufacturer, or inFreesyle CHO-S cells (CHO) (Life technologies) by using the FreestyleMax reagent (Life technologies) essentially as described by themanufacturer. Soluble proteins were transiently expressed in Expi293cells (Life technologies) by using the ExpiFectamine 293 reagent (Lifetechnologies), essentially as described by the manufacturer.

The Fc fusion proteins (CD37mfEC2-FcHis and CD37EC2-FcHis) were purifiedfrom cell culture supernatant using protein A affinity chromatography.

Immunization of Rabbits

Immunization of rabbits was performed at MAB Discovery GMBH (Neuried,Germany). Rabbits were repeatedly immunized with a mixture ofCD37EC2-FcHis and CD37mfEC2-FcHis or HEK293F cells transientlyexpressing human or mfCD37. The blood of these animals was collected andB lymphocytes were isolated. Using a MAB Discovery proprietary process,single B-cells were sorted into wells of microtiter plates and furtherpropagated. The supernatants of these single B-cells were analyzed forspecific binding to CHO-S cells transiently expressing CD37 (CHO-CD37)and mfCD37 (CHO-mfCD37).

Recombinant Antibody Production

Upon analyzing the primary screening results, primary hits were selectedfor sequencing, recombinant mAb production and purification. Uniquevariable heavy chain (VH) and light chain (VL) encoding regions weregene synthesized and cloned into mammalian expression vectors containingthe human IgG1 constant region encoding sequences (Ig Kappa chain andIgG1 allotype G1m (f) containing an E430G mutation (EU numbering) heavychain). During this process an unfavorable, unpaired cysteine in someantibody light chains was replaced by a serine.

Recombinant chimeric antibodies were produced in HEK 293 cells bytransiently cotransfecting the heavy chain (HC) and light chain (LC)encoding expression vectors using an automated procedure on a TecanFreedom Evo platform. Immunoglobulins were purified from the cellsupernatant using affinity purification (Protein A) on a Dionex Ultimate3000 HPLC system.

The reactivity of the produced chimeric (VH rabbit, Fc human) monoclonalantibodies (mAbs) containing a mutation E430G was re-analyzed forbinding to CHO-CD37 or CHO-mfCD37 cells. In addition, binding to thehuman lymphoma cell line Daudi and functionality in the CDC assay onDaudi cells was analyzed.

Example 2: Humanization of Rabbit Chimeric Antibodies

Generation of Humanized Antibody Sequences

Humanized antibody sequences from rabbit antibodiesrabbit-anti-CD37-004, -005, -010 and -016 were generated at Antitope(Cambridge, UK). Humanized antibody sequences were generated usinggermline humanization (CDR-grafting) technology. Humanized V regiongenes were designed based upon human germline sequences with closesthomology to the VH and Vκ amino acid sequences of the rabbit and murineantibodies. A series of four to six VH and four or five Vκ (VL) germlinehumanized V-region genes were designed for each of the rabbitantibodies.

Structural models of the rabbit antibody V regions were produced usingSwiss PDB and analyzed in order to identify amino acids in the V regionframeworks that may be important for the binding properties of theantibody. These amino acids were noted for incorporation into one ormore variant CDR-grafted antibodies.

The heavy and light chain V region amino acid sequence were comparedagainst a database of human germline V and J segment sequences in orderto identify the heavy and light chain human sequences with the greatestdegree of homology for use as human variable domain frameworks. Thegermline sequences used as the basis for the humanized designs are shownin Table 2.

TABLE 2 Closest matching human germline V segment and J segmentsequences. Heavy chain Light chain (K) Human V Human J Human V Human Jregion region region region Rabbit anti- germline germline germlinegermline CD37- segment segment segment segment 004 IGHV3-53*04 IGH34IGKV1-5*01 IGK34 005 IGHV3-53*04 IGH34 IGKV1-12*01 IGK34 010 IGHV3-53*04IGH34 IGKV1-5*03 IGKJ4 016 IGHV3-53*04 IGH34 IGKV1-12*01 IGKJ4

A series of humanized heavy and light chain V regions were then designedby grafting the CDRs onto the frameworks and, if necessary, byback-mutating residues which may be critical for the antibody bindingproperties, as identified in the structural modelling, to rabbitresidues. Variant sequences with the lowest incidence of potential Tcell epitopes were then selected using Antitope's proprietary in silicotechnologies, iTope™ and TCED™ (T Cell Epitope Database) (Perry, L. C.A, Jones, T. D. and Baker, M. P. New Approaches to Prediction of ImmuneResponses to Therapeutic Proteins during Preclinical Development (2008).Drugs in R&D 9 (6): 385-396; Bryson, C. J., Jones, T. D. and Baker, M.P. Prediction of Immunogenicity of Therapeutic Proteins (2010). Biodrugs24 (1):1-8). Finally, the nucleotide sequences of the designed variantshave been codon-optimized.

For antibody IgG1-016-H5L2 a variant with a point mutation in thevariable domain was generated to replace a free cysteine:IgG1-016-H5L2-LC90S (also generated with additional F405L and E430Gmutations). This mutant was generated by gene synthesis (Geneart).

The variable region sequences of the humanized CD37 antibodies are shownin the Sequence Listing herein and in Table 1 above.

Example 3: Generation of Bispecific Antibodies

Bispecific IgG1 antibodies were generated by Fab-arm-exchange undercontrolled reducing conditions. The basis for this method is the use ofcomplementary CH3 domains, which promote the formation of heterodimersunder specific assay conditions as described in WO2011/131746. The F405Land K409R (EU numbering) mutations were introduced in CD37 antibodies tocreate antibody pairs with complementary CH3 domains. The F405L andK409R mutations were in certain cases combined with E430G mutation.

To generate bispecific antibodies, the two parental complementaryantibodies, each antibody at a final concentration of 0.5 mg/mL, wereincubated with 75 mM 2-mercaptoethylamine-HCl (2-MEA) in a total volumeof 100 μL TE at 31° C. for 5 hours. The reduction reaction was stoppedby removing the reducing agent 2-MEA using spin columns (Microconcentrifugal filters, 30k, Millipore) according to the manufacturer'sprotocol.

Example 4: Expression Constructs for Antibodies, Transient Expressionand Purification

For antibody expression the VH and VL sequences were cloned inexpression vectors (pcDNA3.3) containing, in case of the VH, therelevant constant heavy chain (HC), in certain cases containing a F405Lor K409R mutation and/or an E345R or E430G mutation, and, in case of theVL, light chain (LC) regions.

Antibodies were expressed as IgG1,κ. Plasmid DNA mixtures encoding bothheavy and light chains of antibodies were transiently transfected inExpi293F cells (Life technologies, USA) using 293fectin (Lifetechnologies) essentially as described by Vink et al. (Vink et al.,Methods, 65 (1), 5-102014). Next, antibodies were purified byimmobilized protein G chromatography.

The following antibodies were used in the examples:

Wild-Type IgG1 Antibodies:

IgG1-004-H5L2 (having the VH and VL sequences set forth in SEQ ID NO:1and SEQ ID NO:5)

IgG1-005-H1L2 (having the VH and VL sequences set forth in SEQ ID NO:8and SEQ ID NO:12)

IgG1-010-H5L2 (having the VH and VL sequences set forth in SEQ ID NO:15and SEQ ID NO:19)

IgG1-016-H5L2 (having the VH and VL sequences set forth in SEQ ID NO:22and SEQ ID NO:26)

IgG1-G28.1 (having the VH and VL sequences set forth in SEQ ID NO:39 andSEQ ID NO:43-based on SEQ ID No 1 and 3 in EP2241577)

IgG1-G28.1-K409R-delK (also containing a C-terminal heavy chain mutation445-PG-446)

IgG1-37.3 (having the VH and VL sequences set forth in SEQ ID NO:46 andSEQ ID NO:50-based on SEQ ID No 55 and 72 in WO2011/112978)

IgG1-b12 ((having the VH and VL sequences set forth in SEQ ID NO:32 andSEQ ID NO:36-based on the gp120 specific antibody b12 [Barbas, C F. JMol Biol. 1993 Apr. 5; 230(3):812-23])

IgG1 Antibodies with Fc-Fc Interaction-Enhancing Mutation E430G:

IgG1-004-H5L2-E430G

IgG1-005-H1L2-E430G

IgG1-010-H5L2-E430G

IgG1-016-H5L2-E430G

IgG1-G28.1-E430G

IgG1-37.3-E430G

IgG1-b12-E430G

IgG1-005-H1L2-K409R-E430G

IgG1-010-H5L2-K409R-E430G

IgG1-016-H5L2-F405L-E430G

IgG1-016-H5L2-LC90S-F405L-E430G

IgG1-004-E430G

IgG1-005-E430G

IgG1-010-E430G

IgG1-016-E430G

IgG1 Antibodies with Fc-Fc Interaction-Enhancing Mutation E430S:

IgG1-010-H5L2-K409R-E430S

IgG1-016-H5L2-F405L-E430S

IgG1 Antibodies with Fc-Fc Interaction Enhancing Mutation E345K:

IgG1-010-H5L2-K409R-E345K

IgG1-016-H5L2-F405L-E345K

IgG1 Antibodies with Fc-Fc Interaction Enhancing Mutation E345R:

IgG1-G28.1-E345R

IgG1-b12-E345R

IgG1-010-H5L2-K409R-E345R

IgG1-016-H5L2-F405L-E345R

Bispecific Antibodies

bsIgG1-016-H5L2-F405LxIgG1-IgG1-005-H1L2-K409R

bsIgG1-016-H5L2-F405LxIgG1-010-H5L2-K409R

Bispecific Antibodies with Fc-Fc Interaction Enhancing Mutation E430G:

bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G

bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G

bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G

bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G

bsIgG1-b12-F405L-E430Gx005-H1L2-K409R-E430G

IgG1 antibody with FcγR-interaction enhancing mutation 5239D-I332E:

IgG1-G28.1-5239D-I332E

Example 5: Introduction of an Fc-Fc Interaction Enhancing Mutation intoCD37 Antibodies Results in Enhanced, De Novo Capacity to InduceComplement Dependent Cytotoxicity (CDC)

Determination of Complement Dependent Cytotoxicity (CDC)

In a first experiment, tumor cells derived from an untreated CLL patient(AllCells, California, USA), were resuspended in RPMI containing 0.2%BSA (bovine serum albumin) and plated into polystyrene 96-wellround-bottom plates (Greiner bio-one Cat #650101) at a density of0.2×105 cells/well (40 μL/well) and 40 μL of a concentration series ofIgG1-G28.1-K409R-delK, IgG1-G28.1-6345R or IgG1-b12-6345R (0.003-10μg/mL final antibody concentration). IgG1-b12-E345R (based on the gp120specific antibody b12 [Barbas, CF. J Mol Biol. 1993 Apr. 5;230(3):812-23]) was used as negative control. For IgG1-G28.1-K409R-delK,it should be noted that the K409R mutation has no effect on bindingcapacity or capacity to induce CDC. Similarly, the delK (445-PG-446)mutation, which had been introduced into the antibody to facilitatebiochemical analysis, did not affect target binding or capacity toinduce CDC (see below).

After incubation (RT, 10 min while shaking), 20 μL of pooled normalhuman serum (NHS Cat #M0008 Sanquin, Amsterdam, The Netherlands) wasadded to each well as a source of complement and plates were incubatedat 37° C. for 45 minutes. The reaction was stopped by cooling the plateson ice. Next, propidium iodide (PI; 10 μL of a 10 μg/mL solution;Sigma-Aldrich Chemie B. V., Zwijndrecht, The Netherlands) was added andlysis was detected by measurement of the percentage of dead cells(corresponding to PI-positive cells) by flow cytometry (FACS Canto II;BD Biosciences). Graphs were generated using best-fit values of anon-linear dose-response fit with log-transformed concentrations inGraphPad Prism V6.04 software (GraphPad Software, San Diego, Calif.,USA).

In a second experiment, tumor cells from another untreated CLL patient(AllCells, California, USA) were resuspended in RPMI containing 0.2%BSA, were plated into polystyrene 96-well round-bottom plates (Greinerbio-one Cat #650101) at a density of 0.5×105 cells/well (30 μL/well) and50 μL of a concentration series of IgG1-G28.1, IgG1-G28.1-6430G orIgG1-b12 was added (0.003-10 μg/mL final antibody concentration in 3.33×serial dilutions). After incubation (RT, 15 min), 20 μL of pooled normalhuman serum (NHS Cat #M0008 Sanquin, Amsterdam, The Netherlands) wasadded to each well as a source of complement and plates were incubatedat 37° C. for 45 minutes. The reaction was stopped by cooling the plateson ice. Next, propidium iodide (PI; 20 μL of a 10 μg/mL solution;Sigma-Aldrich Chemie B. V., Zwijndrecht, The Netherlands) was added andlysis was detected by measurement of the percentage of dead cells(corresponding to PI-positive cells) by flow cytometry (FACS Canto II;BD Biosciences). Graphs were generated using best-fit values of anon-linear dose-response fit with log-transformed concentrations inGraphPad Prism V6.04 software (GraphPad Software, San Diego, Calif.,USA).

FIGS. 1A and B show that CD37 antibody G28.1 without the Fc-Fcinteraction enhancing E345R or E430G mutation (IgG1-G28.1 orIgG1-G28.1-K409R-delK) did not induce CDC on primary tumor cells fromCLL patients, whereas G28.1 with the Fc-Fc interaction enhancingmutations E345R or E430G (IgG1-G28.1-E345R or IgG1-G28.1-E430G) inducedprofound, dose-dependent CDC of primary CLL cells.

Quantitative Determination of Cell Surface Antigens by Flow Cytometry(Qifi)

The CD37 and membrane complement regulatory proteins (mCRP; CD46, CD55and CD59) expression levels on CLL tumor cells were determined using theHuman IgG Calibrator Kit (Biocytix Cat #CP010). Briefly, tumor cellsderived from a CLL patient (as in first experiment described above),resuspended in RPMI containing 0.2% BSA, were plated into polystyrene96-well round-bottom plates (Greiner bio-one Cat #650101) at a densityof 0.5×105 cells/well (30 μL/well), centrifuged and 50 μL of CD37(Abcam, cat. no. 76522) or control mouse antibody (Purified Mouse IgG1,κIsotype Control, Clone MOPC-21; BD cat. no. 555746) was added. Afterincubation (4° C., 30 min), 50 μL of calibration beads were added intoseparate wells. After washing the beads and cells twice (150 μL FACSbuffer, centrifuging for 3 minutes at 300×g at 4° C. in between washsteps), 50 μL/well secondary antibody (FITC-conjugated) dilution, asprovided in the Human IgG Calibrator Kit, was added. After incubation inthe dark (4° C., 45 min) cells were washed twice with FACS buffer andcells were resuspended in 35 μL FACS buffer and analyzed by flowcytometry (Intellicyt iQue™ screener). The antigen quantity wasdetermined by calculating the antibody-binding capacity based on thecalibration curve, according to the manufacturer's guidelines.

FIG. 2 shows that CD37 was highly expressed on primary tumor cells fromthis CLL patient. The patient showed normal expression levels of mCRP's.

Example 6: Binding of CD37 Antibodies and Variants Thereof to CellSurface Expressed CD37

Binding to cell surface expressed CD37 (Daudi cells, CHO cellsexpressing cynomolgus CD37) was determined by flow cytometry. Cells,resuspended in RPMI containing 0.2% BSA, were seeded at 100,000cells/well in polystyrene 96 well round-bottom plates (Greiner bio-oneCat #650101) and centrifuged for 3 minutes at 300×g, 4° C. Serialdilutions (0.003-10 μg/mL final antibody concentration in 3.33× serialdilutions) of CD37 or control antibodies were added and cells wereincubated for 30 minutes at 4° C. Plates were washed/centrifuged twiceusing FACS buffer (PBS/0.1% BSA/0.01% Na-Azide). Next, cells wereincubated for 30 minutes at 4° C. with R-Phycoerythrin (PE)-conjugatedgoat-anti-human IgG F(ab′)2 (Jackson ImmunoResearch Laboratories, Inc.,West Grove, Pa.; cat #: 109-116-098) diluted 1/100 in PBS/0.1% BSA/0.01%Na-Azide. Cells were washed/centrifuged twice using FACS buffer,resuspended in 30 μL FACS buffer and analyzed by determining meanfluorescent intensities using an Intellicyt iQue™ screener (Westburg).Binding curves were generated using non-linear regression (sigmoidaldose-response with variable slope) analyses within GraphPad Prism V6.04software (GraphPad Software, Sand Diego, Calif., USA).

Binding to Daudi Cells

FIG. 3 shows that humanized CD37 antibodies IgG1-004-H5L2,IgG1-005-H1L2, IgG1-010-H5L2 and IgG1-016-H5L2 showed dose-dependentbinding to Daudi cells. Introduction of the Fc-Fc interaction enhancingE430G mutation, and for IgG1-005-H1L2 also the K409R mutation, intothese antibodies did not affect the binding.

FIG. 4 shows that introduction of the E430G mutation into IgG1-G28.1 orIgG1-37.3 did not affect the binding to Daudi cells.

For antibody IgG1-016-H5L2 a variant with a point mutation in thevariable domain was generated to replace a free cysteine in the lightchain: IgG1-016-H5L2-LC90S. This variant was also generated withadditional F405L and E430G mutations that were previously shown to notaffect target binding characteristics. FIG. 5 shows that theIgG1-016-H5L2, IgG1-016-H5L2-E430G, IgG1-016-H5L2-F405L-E430G andIgG1-016-H5L2-LC90S-F405L-E430G all showed comparable binding to Daudicells, thus that the LC90S mutation did not affect binding.

Binding to CHO Cells Expressing Cynomolgus Monkey CD37

Binding to CHO cells expressing cynomolgus monkey CD37 was determined byflow cytometry using a method as described above. FIG. 6 shows thatIgG1-004-H5L2-E430G, IgG1-005-H1L2-E430G, IgG1-010-H5L2-E430G andIgG1-016-H5L2-E430G showed dose-dependent binding to CHO cellsexpressing cynomolgus monkey CD37. IgG1-G28.1 and IgG1-28.1-E430G didnot bind to CHO cells expressing cynomolgus CD37.

Example 7: Identification of CD37 Antibodies that do not Compete forBinding to CD37

(Lack of) Binding Competition—Determined by Flow Cytometry

CD37 antibodies were labeled with Alexa Fluor 488 NHS Ester(Succinimidyl Ester). 1 mg of CD37 antibody (dissolved in PBS) wastransferred to a 1 ml micro-centrifuge vial (reaction vial). The pH wasraised by addition of a 10% volume of 1 M sodium bicarbonate buffer (pH9). Immediately before use, 1 mg Alexa Fluor 488 NHS Ester (adjusted toroom temperature) was dissolved in 100 μL DMSO. The labeling reactionwas initiated by addition of 10 μL of the fresh Alexa dye solution permg antibody. Reaction vials were capped and mixed gently by inversion.After 1 hour incubation at room temperature, the reaction was quenchedby addition of 50 μL 1M Tris to each reaction vial. Unreacted dye wasremoved from the Alexa-labeled antibody by gel filtration using BioRadPDP10 columns equilibrated with borate saline buffer, according to themanufacturer's directions. Alexa-labeled antibodies were stored at 4° C.and protected from light.

Binding competition between different CD37 antibodies was determined byflow cytometry. Raji cells (ATCC, CCL-86) were resuspended in Rajimedium (RPMI 1640, 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin,10 mM HEPES and 1 mM pyruvate) at a concentration of 1×107 cells/mL.Next, 30 μL aliquots of the cell suspension were transferred into FACStubes together with 30 μL aliquots (40 μg/mL final concentration) ofunlabeled antibody solutions. The mixture was incubated at 37° C. for 15min while shaking gently. Next, A488-labeled antibody dilutions wereprepared and after incubation, 10 μL of the labeled antibodies (4 μg/mLfinal antibody concentration) was transferred to the FACS tubescontaining the unlabeled antibodies and cells. The mixture was incubatedat 37° C. for 15 min while shaking gently. After incubation, sampleswere quenched by adding 4 mL of ice cold PBS, centrifuged for 3 min at4° C. at 2000 rpm, aspirated twice and subsequently resuspended in 125μL of PBS. Binding competition was analyzed by determining meanfluorescent intensities using a BD FACSCalibur (BD Biosciences).Fluorescence intensities were converted to Molecules of EquivalentSoluble Fluorochome (MESF) for quantitation.

FIG. 7A and FIG. 8 show that pre-incubation of Raji cells withIgG1-005-H1L2-E430G and IgG1-010-H5L2-E430G blocked subsequent bindingof IgG1-005-H1L2-E430G and IgG1-010-H5L2E430G, but not ofIgG1-37.3-E430G, IgG1-G28.1-E430G, IgG1-004-H5L2-E430G andIgG1-016-H5L2-E430G.

Pre-incubation of Raji cells with IgG1-004-H5L2-E430G substantiallyreduced subsequent binding of IgG1-37.3-E430G, IgG1-G28.1-E430G,IgG1-004-H5L2-E430G and IgG1-016-H5L2-E430G, but not ofIgG1-005-H1L2-E430G and IgG1-010-H5L2-E430G.

Pre-incubation of Raji cells with IgG1-016-H5L2-E430G blocked subsequentbinding of IgG1-37.3-E430G, IgG1-G28.1-E430G, IgG1-004-H5L2-E430G andIgG1-016-H5L2-E430G, but not of IgG1-005-H1L2-E430G andIgG1-010-H5L2-E430G.

Pre-incubation of cells with IgG1-37.3-E430G blocked the subsequentbinding of all tested antibodies. However, as discussed abovepre-incubating with either of IgG1-005-H1L2-E430G or IgG1-010-H5L2-E430Gdid not block the binding of IgG1-37.3-E430G.

Pre-incubation of cells with IgG1-G28.1-E430G blocked the subsequentbinding of IgG1-37.3-E430G, IgG1-G28.1-E430G, IgG1-004-H5L2-E430G andIgG1-016-H5L2-E430G, but not of IgG1-005-H1L2-E430G andIgG1-010-H5L2-E430G.

(Lack of) Binding Competition—Determined by Functional Screening Using aCDC Assay

To determine whether non-cross-blocking CD37 antibodies show enhancedCDC when combined, and to confirm the potential to functionally combinenon-cross-blocking CD37 antibodies, a CDC assay using individual CD37antibodies and combinations thereof was performed.

Raji cells, resuspended in RPMI containing 0.2% BSA, were plated intopolystyrene 96-well round-bottom plates (Greiner bio-one Cat #650101) ata density of 1×10⁵ cells/well (30 μL/well) and 50 μL of humanized CD37antibodies, variants thereof, combinations thereof or control antibodyIgG1-b12 was added (10 μg/mL final antibody concentration, combinations5+5 μg/mL). After incubation (RT, 15 min, while shaking), 20 μL ofpooled normal human serum (NHS Cat #M0008 Sanquin, Amsterdam, TheNetherlands) was added to each well and plates were incubated at 37° C.for 45 minutes. Plates were centrifuged (3 minutes, 1200 rpm) andsupernatant was discarded. Propidium iodide (PI; 30 μL of a 1.67 μg/mLsolution; Sigma-Aldrich Chemie B.V., Zwijndrecht, The Netherlands) wasadded and lysis was detected by measurement of the percentage of deadcells (corresponding to PI-positive cells) by flow cytometry (IntellicytiQue™ screener, Westburg). Data was analyzed using GraphPad Prismsoftware (Graphpad software, San Diego, Calif., USA).

FIGS. 7B and C show that the combination of IgG1-004-H5L2 plusIgG1-010-H5L2 (with or without E430G mutation) and the combination ofIgG1-005-H1L2 plus IgG1-016-H5L2 (with or without E430G mutation inducedenhanced CDC compared to their individual counterparts. The combinationof IgG1-004-H5L2 plus IgG1-016-H5L2 (with or without E430G mutation) didnot induce enhanced CDC compared to their individual counterparts.

FIGS. 7D and E show that the combination of IgG1-004-H5L2 plusIgG1-005-H1L2 (with or without E430G mutation) and the combination ofIgG1-010-H5L2 plus IgG1-016-H5L2 (with or without E430G mutation inducedenhanced CDC compared to their individual counterparts. The combinationof IgG1-005-H1L2 plus IgG1-010-H5L2 (with or without E430G mutation) didnot induce enhanced CDC compared to their individual counterparts.

FIGS. 7F and G show that the combination of IgG1-37.3 plus IgG1-005-H1L2(with or without E430G mutation) and the combination of IgG1-37.3 plusIgG1-010-H5L2 (with or without E430G mutation induced enhanced CDCcompared to their individual counterparts.

Hence, functional combination studies confirmed the results of thebinding competition studies for described CD37 antibodies and showedthat non-cross-blocking CD37 antibodies can functionally be combined.

Example 8: Introducing an Fc-Fc Interaction Enhancing Mutation intoHumanized CD37 Antibodies Results in Enhanced, De Novo Capacity toInduce Complement Dependent Cytotoxicity (CDC)

Daudi cells, resuspended in RPMI containing 0.2% BSA, were plated intopolystyrene 96-well round-bottom plates (Greiner bio-one Cat #650101) ata density of 1×10⁵ cells/well (30 μL/well) and 50 μL of a concentrationseries of humanized CD37 antibodies and variants thereof, or controlantibody IgG1-b12, was added (0.003-10 μg/mL final antibodyconcentration in 3.33× serial dilutions). After incubation (RT, 15 min),20 μL of pooled normal human serum (NHS, Cat #M0008 Sanquin, Amsterdam,The Netherlands) was added to each well and plates were incubated at 37°C. for 45 minutes. Plates were centrifuged (3 minutes, 1200 rpm) andsupernatant was discarded. Propidium iodide (PI; 30 μL of a 1.67 μg/mLsolution; Sigma-Aldrich Chemie B.V., Zwijndrecht, The Netherlands) wasadded and lysis was detected by measurement of the percentage of deadcells (corresponding to PI-positive cells) by flow cytometry (IntellicytiQue™ screener, Westburg). Graphs were generated using best-fit valuesof a non-linear dose-response fit with log-transformed concentrations inGraphPad Prism V6.04 software (GraphPad Software, San Diego, Calif.,USA).

FIG. 9 shows that IgG1-004-H5L2, IgG1-005-H1L2, IgG1-010-H5L2 andIgG1-016-H5L2 did not induce CDC in Daudi cells. Upon introduction ofthe Fc-Fc interaction enhancing E430G mutation, these antibodies(IgG1-004-H5L2-E430G, IgG1-005-H1L2-E430G, IgG1-010-H5L2-E430G andIgG1-016-H5L2-E430G) induced profound, dose-dependent CDC of Daudicells.

FIG. 10A shows that IgG1-G28.1 and IgG1-37.3 did not induce CDC on Daudicells. Upon introduction of the Fc-Fc interaction enhancing E430Gmutation, these antibodies (IgG1-G28.1-E430G and IgG1-37.3-E430G)induced profound, dose-dependent CDC of Daudi cells.

For antibody IgG1-016-H5L2 a variant with a point mutation in thevariable domain was generated to replace a free cysteine in the lightchain: IgG1-016-H5L2-LC90S. In addition, this variant was also generatedwith an F405L mutation (previously shown not to affect target binding orCDC) and an Fc-Fc interaction enhancing E430G mutation. FIG. 11 showsthat the IgG1-016-H5L2-E430G, IgG1-016-H5L2-F405L-E430G andIgG1-016-H5L2-LC90S-F405L-E430G all showed comparable activity in an invitro CDC assay, thus that the LC90S mutation did not affect thecapacity to induce CDC. IgG1-016-H5L2 did not induce CDC on Daudi cells.

Also, introduction of other Fc-Fc interaction enhancing mutations,E345K, E345R, E430S and RRGY, in IgG1-010-H5L2 and IgG1-016-H5L2resulted in profound CDC of Daudi cells. FIGS. 10B and C show thatmaximum lysis of Daudi cell was comparable for all tested Fc-Fcinteraction enhancing mutations.

Example 9: Bispecific CD37 Antibodies with an Fc-Fc InteractionEnhancing Mutation are More Potent in Inducing CDC than MonospecificBivalent CD37 Antibodies with an Fc-Fc Interaction Enhancing MutationDue to Monovalent Binding and Dual Epitope Targeting

F405L or K409R mutations were introduced into humanized CD37 antibodiescontaining the E430G mutation, to allow for the generation of bispecificantibodies (bsIgG1) with two CD37-specific Fab-arms that do not competefor binding to CD37. The capacity of bispecific CD37 antibodiescontaining the E430G mutation to induce CDC was determined as describedabove, and compared to that of CD37 monospecific bivalent antibodiescontaining the E430G mutation, a combination of two CD37 monospecificbivalent antibodies containing the E430G mutation that do not competefor binding to CD37 (with the end concentration of the combinedantibodies together identical to the concentration of the individualbispecific antibodies), monovalent CD37 antibodies containing the E430Gmutation (i.e. bispecific antibodies containing one CD37-specific Fabarm and one non-binding Fab-arm derived from IgG1-b12, and containingthe E430G mutation) or a combination of two monovalent CD37 antibodiescontaining the E430G mutation that do not compete for binding to CD37.

CDC on Daudi Cells

FIG. 12A shows thatbsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G was more potentthan either IgG1-005-H1L2-E430G or IgG1-016-H5L2-E430G in inducing CDCon Daudi cells. The bispecificbsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G was also morepotent than a combination of IgG1-005-H1L2-K409R-E430G plusIgG1-016-H5L2-F405L-E430G. Monovalent CD37-binding antibodiesbsIgG1-b12-F405L-E430Gx005-H1L2-K409R-E430G andbsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G also induced CDC onDaudi cells, but were less efficient in doing so thanbsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G.

FIG. 12B shows thatbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G was more potentthan either IgG1-010-H5L2-E430G or IgG1-016-H5L2-E430G in inducing CDCon Daudi cells. The bispecificbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G was also morepotent than a combination of IgG1-010-H5L2-E430G plusIgG1-016-H5L2-E430G. Monovalent binding antibodiesbsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G andbsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G also induced CDC on Daudicells, with bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G being lesspotent and bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G beingequally potent compared tobsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G.

The capacity to induce CDC by bispecific CD37 antibodies containing theE430G mutation was also compared to that of bispecific CD37 antibodieswithout the E430G mutation. FIG. 13 shows thatbsIgG1-016-H5L2-F405Lx005-H1L2-K409R as well asbsIgG1-016-H5L2-F405Lx010-H5L2-K409R were capable of inducing CDC onDaudi cells, but were less potent in doing so compared to their E430Gcontaining counterpartsbsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G andbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G.

CDC on OCI-Ly-7 Cells

FIG. 12C shows that monovalent binding antibodiesbsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G andbsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G were more potent in inducingCDC on OCI-Ly-7 cells compared to their monospecific bivalent bindingcounterparts, IgG1-016-H5L2-E430G and IgG1-010-H5L2-E430G. Thecombination of monovalent binding antibodies(bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G plusbsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G) was more potent than thecombination of bivalent antibodies (IgG1-010-H5L2-E430G plusIgG1-016-H5L2-E430G), as demonstrated by a consistent lower EC50 in twoindependent experiments (FIG. 12D). Also,bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G was more potentin inducing CDC on OCI-Ly-7 cells than the combination of bivalentantibodies (IgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G), asdemonstrated by a consistent lower EC50 in three independent experiments(FIG. 12E).

The potency of the combination of monovalent binding antibodies(bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G plusbsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G) and ofbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G in inducing CDCin OCI-Ly-7 cells was comparable.

CDC on Primary CLL Tumor Cells

The capacity of bispecific CD37 antibodies containing the E430G mutationto induce CDC on tumor cells derived from a CLL patient was determinedas described above, and compared to that of CD37 antibodies containingthe E430G mutation or a combination of CD37 antibodies containing theE430G mutation or monovalent CD37 antibodies containing the E430Gmutation.

FIG. 14A shows thatbsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G was more potentthan either IgG1-005-H1L2-K409R-E430G or IgG1-016-H5L2-F405L-E430G ininducing CDC on primary CLL tumor cells. The bispecificbsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G was also morepotent than a combination of IgG1-005-H1L2-K409R-E430G plusIgG1-016-H5L2-F405L-E430G. Monovalent binding antibodiesbsIgG1-b12-F405L-E430Gx005-H1L2-K409R-E430G andbsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G also induced CDC onprimary CLL tumor cells, but were less efficient in doing so thanbsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G.

FIG. 14B shows thatbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G was more potentthan either IgG1-010-H5L2-E430G or IgG1-016-H5L2-E430G in inducing CDCon primary CLL tumor cells. The bispecificbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G was also morepotent than a combination of IgG1-010-H5L2-E430G plusIgG1-016-H5L2-E430G. Monovalent binding antibodiesbsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G andbsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G also induced CDC on primaryCLL tumor cells, with bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G beingless potent and bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G beingequally potent compared tobsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G.

Example 10: Bispecific CD37 Antibodies with an Fc-Fc InteractionEnhancing Mutation Induce CDC on a Variety of B Cell Lymphoma Cell Lineswith a Wide Range of CD37 Expression

The capacity of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G,at a concentration of 10 μg/mL, to induce CDC was determined (asdescribed above) on a range of B cell lymphoma cell lines, derived froma variety of B cell lymphoma subtypes. The expression levels of CD37molecules on the cell surface of these cell lines were determined byquantitative flow cytometry as described above.

Table 3 gives an overview of the cell lines tested.

TABLE 3 B cell lymphoma cell lines. Cell line Lymphoma type Source JVM-2MCL DSMZ; ACC 12 JVM-13 MCL ATCC; CRL-3003 Jeko-1 MCL DSMZ; ACC 553Z-138 MCL ATCC; CRL-3001 Daudi Burkitt's ATCC; CCL-213 Raji Burkitt'sATCC; CCL-86 Wien-133 Burkitt's BioAnaLab, Oxford, U.K SU-DHL-8 DLBCLDSMZ; ACC 573 OCI-Ly19 DLBCL DSMZ; ACC 528 OCI-Ly7 DLBCL DSMZ; ACC 688SU-DHL-4 DLBCL DSMZ; ACC 495 RC-K8 DLBCL DSMZ; ACC 561 U-2932 DLBCLDSMZ; ACC 633 WIL-25 Plasmablastic ATCC; CRL-8885 RI-1 DLBCL DSMZ; ACC585 WSU-DLCL2 DLBCL DSMZ; ACC 575

FIG. 15 shows thatbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G induced CDC on awide range of B cell lymphoma cell lines, derived from various B celllymphoma types.

Example 11: Bispecific CD37 Antibodies with an Fc-Fc InteractionEnhancing Mutation are More Potent in Inducing Antibody-DependentCell-Mediated Cytotoxicity (ADCC)

Labeling of Target Cells

The capacity of CD37 antibodies to induce ADCC was determined by achromium release assay. Daudi or Raji cells were collected (5×10⁶cells/mL) in 1 mL culture medium (RPMI 1640 supplemented with 10% DonorBovine Serum with Iron (DBSI; ThermoFischer, Cat #10371029) andPenicillin Streptomycin mixture (pen/strep), to which 100 μCi 51Cr(Chromium-51; PerkinElmer, Cat #NEZ030005MC) had been added. Cells wereincubated in a water bath at 37° C. for 1 hour while shaking. Afterwashing of the cells (twice in PBS, 1500 rpm, 5 min), the cells wereresuspended in RPMI 1640/10% DBSI/pen/strep and counted by trypan blueexclusion. Cells were diluted to a density of 1×10⁵ cells/mL.

Preparation of Effector Cells

Peripheral blood mononuclear cells from healthy volunteers (Sanquin,Amsterdam, The Netherlands) were isolated from 45 mL of freshly drawnheparin blood (buffy coats) by Ficoll density centrifugation (BioWhittaker; lymphocyte separation medium, cat 17-829E) according to themanufacturer's instructions. After resuspension of cells in RPMI1640/10% DBSI/pen/strep, cells were counted by trypan blue exclusion anddiluted to a density of 1×107 cells/mL.

ADCC Assay Procedure

50 μL of ⁵¹Cr-labeled targets cells were pipetted into 96-wellround-bottom microtiter plates (Greiner Bio-One; Cat #650101), and 50 μLof a concentration series of (1.5-5,000 ng/mL final concentrations in3-fold dilutions) CD37 or control antibodies, diluted in RPMI 1640/10%DBSI/pen/strep was added. Cells were incubated at room temperature (RT)for 15 min and 50 μL effector cells were added, resulting in an effectorto target ratio of 100:1. Cells were incubated for 4 hours at 37° C. and5% CO2. For determination of maximal lysis, 50 μL 51Cr-labeled Daudicells (5.000 cells) were incubated with 100 μL 5% Triton-X100; fordetermination of spontaneous lysis (background lysis), 5,00051Cr-labeledDaudi cells were incubated in 150 μL medium without any antibody oreffector cells. The level of antibody-independent cell lysis wasdetermined by incubating 5,000 Daudi cells with 500,000 PBMCs withoutantibody. Plates were centrifuged (1200 rpm, 10 min) and 25 μL ofsupernatant was transferred to 100 μL Microscint-40 solution (Packard,Cat #6013641) in 96-Wells plates. Plates were sealed and shaken for 15minutes at 800 rpm and released 51Cr was counted using a scintillationcounter (TopCount®, PerkinElmer). The percentage specific lysis wascalculated as follows:% specific lysis=(cpm sample−cpm spontaneous lysis)/(cpm maximallysis−cpm spontaneous lysis) wherein cpm is counts per minute.

FIG. 16A shows thatbsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G was more potentthan either IgG1-005-H1L2-K409R-E430G or IgG1-016-H5L2-F409L-E430G orthan a combination of IgG1-005-H1L2-K409R-E430G plusIgG1-016-H5L2-F405L-E430G in inducing ADCC on Daudi cells.

FIG. 16B shows thatbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G was more potentthan either IgG1-010-H5L2-E430G or IgG1-016-H5L2-E430G or a combinationof IgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G in inducing ADCC onDaudi cells.

FIG. 16C shows similar results as FIG. 16B for PBMCs from a differentdonor, and in addition shows thatbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G was more potentthan monovalent binding antibodiesbsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G andbsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G in inducing ADCC on Rajicells.

Example 12: Bispecific CD37 Antibodies with an Fc-Fc InteractionEnhancing Mutation Induce Potent Ex Vivo CDC in Primary Tumor Cells fromPatients with Various B Cell Malignancies

The CDC efficacy of bsIgG1-016-H5L2-LC90S-F405Lx010-H5L2-K409R-E430G wasanalyzed using primary patient-derived tumor cells from five different Bcell malignancies: chronic lymphocytic leukemia (CLL), follicularlymphoma (FL), diffuse large B cell lymphoma (DLBCL), mantle celllymphoma (MCL) and Non-Hodgkin's lymphoma (not further specified). Allpatient samples were obtained after written informed consent and storedusing protocols approved by the VUmc Medical Ethical Committee inaccordance with the declaration of Helsinki. Patient bone marrowmononuclear cells (BMNCs) or peripheral blood mononuclear cells (PBMCs)were isolated by density-gradient centrifugation (Ficoll-Paque PLUS, GEHealthcare) from bone marrow aspirates or peripheral blood samples ofpatients. Cells were either used directly or stored in liquid nitrogenuntil further use.

Patient lymph node tissue was dissected into small fragments andcollected in a-MEM medium (ThermoFischer Scientific, Waltham, Mass.)containing 1% Penicillin-Streptomycin, 0.2% heparin and 5% plateletlysate and left overnight at 37° C. After incubation, the supernatant(non-stromal cell compartment including tumor cells) was collected andcells were filtered using a 70 μM Easy Strainer (Greiner Bio-one). Cellswere counted, resuspended in RPMI 1640 medium containing 25%heat-inactivated FBS and 10% DMSO, and frozen in liquid nitrogen untilfurther use.

The CD37 and membrane complement regulatory proteins (mCRP; CD46, CD55and CD59) expression levels on isolated patient cells were determinedusing a QifiKit (DAKO, cat. no. K007811). Cells were incubated with thepurified antibodies CD37 (BD, cat. no. 555456), CD46 (BioLegend, cat.no. 352404), CD55 (BioLegend, cat. no. 311302), CD59 (BioLegend, cat.no. 304702), and b12 (Genmab) at 4° C. for 30 min. After this the methodas provided by the QifiKit manufacturer was used. After the final stepof Qifi kit procedure, cells were incubated with lymphoma cell specificmarkers to enable tumor cell identification. FIG. 17 shows theexpression levels per indication.

The patient-derived tumor cells were opsonized with 10 μg/mL or 100μg/mL bsIgG1-016-H5L2-LC90S-F405Lx010-H5L2-K409R-E430G and CDC inductionwas assessed in the presence of 20% pooled NHS. The following cellmarkers were used to identify different cell populations: CD45-KO(Beckman Coulter B36294), CD19-PC7 (Beckman Coulter, cat. no. IM3628),CD3-V450 (BD, cat. no. 560365), CD5-APC (BD, cat. no. 345783), CD5-PE(DAKO, cat. no. R084201), CD10-APC-H7 (BD, cat. no. 655404), CD10-PE(DAKO, cat. no. R084201), CD23-FITC (Biolegend, cat. no. 338505),lambda-APC-H7 (BD, cat. no. 656648), kappa-PE (DAKO, cat. no. R043601)and lambda-FITC (Emelca Bioscience CYT-LAMBF). Within the CD45+ cellpopulation, malignant B cells were defined by different markersdepending on the indication: CD3−/CD19+/CD5+ (CLL), CD3−/CD19+/CD10+(FL, DLBCL), CD3−/CD19+/CD5+/CD23− (MCL). In case malignant B cellscould not be identified based on these markers, malignant cells wereidentified based on clonality using kappa/lambda staining. In a fewsamples, malignant B cells could also not be identified based onclonality; in these cases the total B cell population was assessed,without distinction between normal and malignant B cells. Killing wascalculated as the fraction of 7-amino actinomycin D (7-AAD; BD, cat. no.555816) positive malignant B cells (%) determined by an LSRFortessa flowcytometer (BD Biosciences, San Jose, Calif.).

FIG. 18 shows that bsIgG1-016-H5L2-LC90S-F405Lx010-H5L2-K409R-E430G washighly potent (lysis of more than 50%) in inducing CDC in tumor cellsderived patients with CLL, FL, MCL, DLBCL or B-NHL (not furtherspecified). In cells from one patient with relapsed/refractory FL,bsIgG1-016-H5L2-LC90S-F405Lx010-H5L2-K409R-E430G was less capable ofinducing CDC.

Example 13: Binding of a Bispecific CD37 Antibody with an Fc-FcInteraction Enhancing Mutation to Human or Cynomolgus Monkey B Cells inWhole Blood, and Induction of Cytotoxicity in B Cells in Whole Blood

Binding to Human or Cynomolgus Monkey B Cells

Binding to human or cynomolgus monkey B cells was determined in a wholeblood binding assay. Heparin-treated human blood from healthy volunteerswas derived from UMC Utrecht (Utrecht, The Netherlands), hirudin-treatedblood from cynomolgus monkeys was derived from Covance (Munster,Germany). Blood was aliquoted to wells of a 96-well round-bottom plate(Greiner Bio-one, cat. no. 65010; 35 μL/well). Red blood cells (RBC)were lysed by addition of 100 μL RBC lysis buffer (10 mM KHCO₃ [SigmaP9144], 0.1 mM EDTA [Fluka 03620] and 0.15 mM NH₄CL [Sigma A5666]) andincubated on ice until RBC lysis was complete. After centrifugation for3 minutes at 300×g, cells were incubated for 30 minutes at 4° C. withserial dilutions (0.014-30 μg/mL final antibody concentration in 3×serial dilutions) of Alexa-488 labeledbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G or Alexa-488labeled control IgG1 (IgG1-b12) and a directly labeled antibody toidentify B cells (among a mixture of antibodies to further identifyblood cell subsets):

For human blood B cells the following antibody was used

Target protein Clone Label Target cells Company Cat. no. CD19 HIB19BV711 B cells Biolegend 302245For cynomolgus monkey blood B cells the following antibody was used

Target protein Clone Label Target cells Company Cat. no. CD19 J3-119 PEB cells Beckman A07769 Coulter

Cells were pelleted and washed twice in 150 μL FACS buffer andresuspended in 150 μL TO-PRO-3 (end concentration 0.2 μM; MolecularProbes, cat no. T3605). Samples were measured by flow cytometry using anLSRFortessa flow cytometer. Binding is expressed as geometric mean ofA488 fluorescence intensity for viable TO-PRO-3⁻/CD14⁻/CD19⁺ B-cells(human) or viable TO-PRO-3⁻/CD14⁻/CD19⁺/CD20⁺ B-cells (cynomolgusmonkey). Log-transformed data were analyzed using best-fit values of anon-linear dose-response fit in GraphPad PRISM.

FIG. 19 shows the concentration dependent binding ofbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G to B cells in (A)human and (B) cynomolgus monkey blood, for one representativedonor/animal. The average EC₅₀ values for binding to human andcynomolgus monkey B cells were in the same range ([0.85 μg/mL±0.284based on binding to B cells in blood from 6 human donors] and [0.63μg/mL±0.228 based on binding to B cells in blood from 4 animals],respectively), indicating thatbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G shows comparablebinding to human and cynomolgus monkey CD37.

Cytotoxicity to Human or Cynomolgus Monkey B Cells

Cytotoxicity towards human or cynomolgus monkey B cells was determinedin a whole blood cytotoxicity assay. Hirudin-treated human blood fromhealthy volunteers was derived from UMC Utrecht (Utrecht, TheNetherlands), hirudin-treated blood from cynomolgus monkeys was derivedfrom Covance (Munster, Germany). Blood was aliquoted to wells of a96-well round-bottom plate, 35 μL/well.

Serial dilutions (0.0005-10 μg/mL final antibody concentration in 3×serial dilutions; final volume 100 μL/well) ofbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G or IgG1-b12 wereadded. In cytotoxicity assays using human whole blood, the monoclonalFcγR-interaction enhanced CD37 specific antibody IgG1-G28.1-5239D-I332Ewas included as reference. Samples were incubated at 37° C. for 4 hours.Thereafter, red blood cells were lysed as described above and sampleswere stained to identify B cells as described above. Cells were pelletedand washed twice in 150 μL FACS buffer and resuspended in 150 μLTO-PRO-3 (end concentration 0.2 μM; Molecular Probes, cat no. T3605).Samples were measured by flow cytometry using an LSRFortessa flowcytometer. After exclusion of doublets the percentage viableTO-PRO-3⁻/CD14⁻/CD19⁺ B-cells (human) or viableTO-PRO-3⁻/CD14⁻/CD19⁺/CD20⁺ B cells (cynomolgus monkey) was determined.The percentage B-cell depletion was calculated as follows: % B celldepletion=100*[(% B-cells no Ab control−% B cells sample)/(% B cells noAb control)]. Log-transformed data were analyzed using best-fit valuesof a non-linear dose-response fit in GraphPad PRISM.

FIG. 20 shows the concentration dependent cytotoxicity ofbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G to B cells in (A)human and (B) cynomolgus monkey blood, for one representativedonor/animal.

Based on EC₅₀, the capacity ofbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G to inducecytotoxicity in human and cynomolgus monkey B cells was comparable: theaverage EC50 for cytotoxicity to human B cells (in blood from 6 donors)was 0.077 μg/mL 0.039; the average EC50 for cytotoxicity to cynomolgusmonkey B cells (in blood from 4 animals) was 0.043 μg/mL±0.019.

FIG. 20A also shows the cytotoxicity of the FcγR-interaction enhancedmonoclonal CD37 antibody IgG1-G28.1-S239D-I332E to human B cells for arepresentative responding donor, which showed lower cytotoxicity thanbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G. In B cells from3 responding donors, a maximum B-cell depletion of 50% byIgG1-G28.1-5239D-I332E was measured, whereas in 3 other donors nocytotoxicity to B cells by this antibody was measured.BsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G inducedcytotoxicity in 93-99% of B cells in 6/6 donors. Binding ofIgG1-G28.1-5239D-I332E to CD37 expressed on Daudi cells was comparableto that of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G (datanot shown).

Example 14: Potent CDC Activity by a Combination of a Bispecific CD37Antibody with an Fc-Fc Interaction Enhancing Mutation with aCD20-Specific Antibody

The capacity to induce CDC was tested for a combination ofbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G and an anti-CD20antibody (IgG1-CD20-ofa; ofatumumab) on patient derived CLL tumor cellsobtained from ConversantBio (Huntsville, Ala., USA). Patient derivedPBMCs were resuspended in RPMI containing 0.2% BSA (bovine serumalbumin) and plated into polystyrene 96-well round-bottom plates(Greiner bio-one Cat #650101) at a density of 0.1×10⁶ cells/well (30μL/well) and 50 μL of a concentration series ofbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G (0.0625-0.05μg/mL) and IgG1-CD20-ofa (1-8 μg/mL) was added in 2-fold dilutions.BsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G and IgG1-CD20-ofawere combined at antibody concentrations that were based on relativepotency (differences in EC50s) of each of the antibodies, by mixing twoconcentrations that would, on average, separately reach the same effect.IgG1-b12 was used as negative control.

After incubation (RT, 15 min while shaking), 20 μL of pooled normalhuman serum (NHS Cat #M0008 Sanquin, Amsterdam, The Netherlands) wasadded to each well as a source of complement and plates were incubatedat 37° C. for 45 minutes. The reaction was stopped by cooling the plateson ice. After centrifugation for 3 minutes at 300×g, cells were washedtwice with 150 μL FACS buffer and incubated for 30 minutes at 4° C. withan R-Phycoerythrin (PE) labeled mouse-anti-human IgG1-CD19 antibody(clone J3-119, Beckman Coulter, cat no. A07769, 1:50 diluted from stock)to determine the tumor B cells and TO-PRO-3 (end concentration 0.2 μM;Molecular Probes, cat no. T3605) for the identification of dead cells.Cells were pelleted and washed twice in 150 μL FACS buffer and measuredby flow cytometry using an LSRFortessa flow cytometer. The percentage ofviable cells was calculated as follows: % viable cells=100*(#TO-PRO-3negative events)/(# total events).

FIGS. 21A-D show that bothbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G and ofatumumabinduced CDC in tumor cells derived from 2 CLL patients, with CDCactivity increasing with increasing dose levels. CombiningbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G with ofatumumabresulted in enhanced CDC activity at all tested concentrations for bothCLL patients tested, although these effects were less evident at higherantibody concentrations, where almost complete cell kill was induced bythe single agents (FIGS. 21A and B). These results indicate that theaddition of ofatumumab tobsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G can improveCDC-mediated tumor cell kill in malignant B cells obtained from CLLpatients.

Example 15: Anti-Tumor Activity of a Bispecific CD37 Antibody with anFc-Fc Interaction Enhancing Mutation in Xenograft Models of B CellMalignancies

Anti-Tumor Activity in a Subcutaneous JVM-3 Human Chronic B CellLeukemia Xenograft Model

JVM-3 cells (1×10⁷) were inoculated into the right flank of CB17.SCIDmice and antibody treatment (3 weekly doses of 0.1, 0.3, 1, 3 or 10mg/kg, injected intravenously; IgG1-b12 was used as negative control,dosed at 10 mg/kg) was initiated when tumors reached a mean volume ofapproximately 158 mm³. Tumor volumes were measured twice weekly in twodimensions using a caliper, and the volume was expressed in mm³ usingthe formula: V=(L×W×W)/2, where V is tumor volume, L is tumor length(the longest tumor dimension) and W is tumor width (the longest tumordimension perpendicular to L).

FIG. 22A shows the tumor volume per dose group over time, FIG. 22B showsthe tumor volumes per mouse per dose group on day 25 when all groupswere still complete. Three weekly doses ofbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G at 1, 3 or 10mg/kg significantly reduced the JVM-3 cell tumor growth, whereas dosingat 0.1 or 0.3 mg/kg did not affect tumor growth (Mann Whitney test,p<0.01).

Anti-Tumor Activity in an Intravenous Daudi-Luc Burkitt's LymphomaXenograft Model

On day 0, SCID mice (C.B-17/IcrHan®Hsd-Prkdcscid; Harlan) wereintravenously injected with Daudi-luc cells (luciferase transfectedDaudi cell, 2.5×10⁶ cells/mouse). At day 14, 21 and 28, mice wereinjected intraperitoneally with 0.1, 0.3, 1, 3 or 10 mg/kg ofbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G. IgG1-b12 wasused as negative control antibody, dosed at 10 mg/kg. Tumor growth wasevaluated weekly (starting at day 2) by bioluminescence imaging (BLI).Mice were injected intraperitoneally with 100 μL firefly D-luciferin (30mg/mL; Caliper LifeSciences, cat. no. 119222) and bioluminescence(radiance in p/s/cm²/sr [photons per second per cm² per square radian])was measured under isoflurane anesthesia using a BiospaceBioluminescence Imaging System (PerkinElmer; mice were imaged from thedorsal site).

FIG. 23A shows luciferase activity (bioluminescence, as a measure oftumor volume) per dose group over time, FIG. 23B shows the luciferaseactivity per mouse per dose group on day 36 when all groups were stillcomplete. Three weekly doses ofbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G at 0.1, 0.3, 1, 3or 10 mg/kg significantly reduced the in vivo growth of Daudi-luc cells(One Way Anova, Uncorrected Fisher's LSD).

Example 16: Evaluation of Plasma Clearance of a Bispecific CD37 Antibodywith an Fc-Fc Interaction Enhancing Mutation in SCID Mice

11-12 week old, female SCID mice (C.B-17/IcrHan®Hsd-Prkdcscid; Harlan)(3 mice per group) were injected intravenously (i.v.) injected with asingle dose of 100 μg (5 mg/kg) or 500 μg (25 mg/kg) ofbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G or IgG1-b12. Theexperiment was set up to study antibody clearance in absence oftarget-mediated clearance as neitherbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G nor IgG1-b12 showcross-reactivity with mouse.

50-100 μL blood samples were collected from the saphenous vein at 10minutes, 4 hours, 24 hours, 2 days, 7 or 8 days, 14 days and 21 daysafter antibody administration. Blood was collected into heparincontaining vials and centrifuged for 5 minutes at 10,000 g. Plasmasamples were diluted 1:50 for mice dosed with 5 mg/kg (20 μL sample in980 μL PBSA (PBS supplemented with 0.2% bovine serum albumin (BSA)) and1:20 for mice dosed with 25 mg/kg (20 μL sample in 380 μL PBSA) andstored at −20° C. until determination of mAb concentrations.

Human IgG concentrations were determined using a sandwich ELISA. MousemAb anti-human IgG-kappa clone MH16 (CLB Sanquin, The Netherlands; cat.no. M1268), coated in 100 μL overnight at 4° C. to 96-well MicrolonELISA plates (Greiner, Germany) at a concentration of 2 μg/mL, was usedas capturing antibody. After blocking plates with PBSA for 1 hour atroom temperature (RT), samples were added, serially diluted in PBSA, andincubated on a plate shaker for 1 hour at RT. Plates were washed threetimes with 300 μL PBST (PBS supplemented with 0.05% Tween 20) andsubsequently incubated for 1 hour at RT with goat anti-human IgGimmunoglobulin (Jackson, West Grace, Pa.; cat. no. 109-035-098; 1:10.000in PBST supplemented with 0.2% BSA). Plates were washed again threetimes with 300 μL PBST before incubation with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS; Roche, Mannheim, Germany)protected from light. The reaction was stopped by adding 100 μL 2%oxalic acid. Absorbance was measured in a microplate reader (Biotek,Winooski, Vt.) at 405 nm. Human IgG concentration was calculated byusing the injected material as a reference curve. As a plate control,purified human IgG1 (The binding site, cat. no. BP078) was included.Human IgG concentrations (in μg/mL) were plotted (FIGS. 24A and C) andthe area under the curve (AUC) was calculated using Graphpad prism 6.0.IgG clearance until the last day of blood sampling (day 21) wasdetermined by the formula D*1.000/AUC, in which D is the dose ofinjection (1 mg/kg) (FIGS. 24B and D).

There were no substantial differences between plasma clearance rates ofbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G and IgG1-b12,demonstrating thatbsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G showed acomparable pharmacokinetic profile as wild type human IgG1 in absence oftarget binding.

Example 17: Determination of the Contribution of CD37 Amino AcidResidues to Binding of CD37 Antibodies Using Alanine Scanning

Library Design

A CD37 single residue alanine library was synthesized (Geneart) in whichall amino acid (aa) residues in the extracellular domains of human CD37(Uniprot P11049) were individually mutated to alanines except forpositions already containing alanines or cysteines. Cysteines were notmutated to minimize the chance of structural disruption of the antigen.The library was cloned in the pMAC expression vector containing aCMV/TK-polyA expression cassette, an Amp resistance gene and a pBR322replication origin.

Library Production and Screening

The wild type CD37 and alanine mutants were expressed individually inFreeStyle HEK293 cells according to the manufacturer's instructions(Thermo Scientific). One day post transfection the cells were harvested.Approximately 100,000 cells were incubated with 20 μL Alexa488conjugated bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G (monovalentbinding 010) or Alexa488 conjugatedbsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G (monovalent binding016) at a concentration of 3 μg/mL in FACS buffer (PBS+0.1% (w/v) bovineserum albumin (BSA)+0.02% (w/v) sodium azide). Cells were incubated for1 hour at room temperature. Subsequently, cells were washed twice byadding 150 μL FACS buffer and removing the supernatant aftercentrifugation. Cells were resuspended in 20 μL fresh FACS buffer andstored at 4° C. until analysis by flow cytometry using an iQue screener(IntelliCyt). The entire experiment was performed 2 times.

Data Analysis

For every sample, the average antibody binding per cell was determinedas the geometric mean of the fluorescence intensity (gMFI) for theungated cell population. The gMFI is influenced by the affinity of theantibody for the CD37 mutant and the expression level of the CD37 mutantper cell. Since specific alanine mutations can impact the surfaceexpression level of the mutant CD37, and to correct for expressiondifferences for each CD37 mutant in general, data were normalizedagainst the binding intensity of a non-competing CD37 specific controlantibody (in this example antibodies monovalent binding 010 andmonovalent binding 016 were non-competing antibodies and one antibodywas used as control for the other antibody), using the followingequation:

${{Normalized}\mspace{14mu}{gMFI}_{{aa}\mspace{14mu}{position}}} = {Lo{g_{10}\left( \frac{{gMFI}_{{Test}\mspace{14mu}{Ab}}}{{gMFI}_{{Control}\mspace{14mu}{Ab}}} \right)}}$

In which ‘aa position’ refers to either a particular alanine mutantposition in CD37 or wild type (wt) CD37.

To express loss or gain of binding of the antibodies the standard scorewas determined according to the following calculation:

${{zscore}\left( {{fold}{\mspace{11mu}\;}{change}} \right)} = \frac{{{Normalized}\mspace{14mu}{gMFI}_{{aa}\mspace{14mu}{position}}} - \mu}{\sigma}$

Where μ and σ are the mean and standard deviation (SD) of the NormalizedgMFI of all mutants.

Gain of binding in most cases will be caused by loss of binding of thereference antibody to specific ala mutants. Using these calculations,amino acid positions for which, upon replacing the amino acid withalanine, there is no loss or gain of binding by a particular antibodywill give a zscore of ‘0’, gain of binding will result in ‘zscore>0’ andloss of binding will result in ‘zscore<0’. To correct for samplevariation, only CD37 amino acid residues where the zscore was lower than−1.5 were considered ‘loss of binding mutants’. In case the gMFI of thecontrol antibody for a particular CD37 mutant was lower than the meangMFI-2.5×SD of the mean gMFI_(Control Ab), data were excluded fromanalysis (as for those CD37 mutants it was assumed expression levelswere not sufficient).

FIG. 25 shows the ‘zscore (fold change)’ of the CD37 antibodies to CD37variants with ala mutations at positions 42 to 131 (according to SEQ IDNo 94). The results indicate that:

-   -   binding of antibody 010 is at least dependent on aa Y182, D189,        T191, I192, D194, K195, V196, I197 and P199 of human CD37,    -   binding of antibody 016 is at least dependent on aa E124, F162,        Q163, V164, L165 and H175 of human CD37.        In Summary

In summary, bispecific antibodies composed of two CD37-specificantibodies that do not compete for target binding with an Fc-Fcinteraction enhancing mutation, showed the most favorable combination ofCDC potency and ADCC potency in CD37-positive tumor cells. For botheffector mechanisms, the bispecific antibodies with the Fc-Fcinteraction enhancing mutation showed superior potency compared to thecombination of two non-competing CD37 antibodies containing the Fc-Fcinteraction enhancing mutation or to the single CD37 antibodies with theFc-Fc interaction enhancing mutation.

Example 18: In Vitro Evaluation of CDC Activity of Mixtures of NovelHexamerization-Enhanced CD37 Antibodies with Clinically Established CD20Antibody Products on Raji Cells

The CDC activity of mixtures of CD37 antibodies with an Fc-Fcinteraction enhancing mutation, IgG1-37.3-E430G, IgG1-G28.1-E430G,IgG1-004-E430G, IgG1-005-E430G, IgG1-010-E430G and IgG1-016-E430G (thelatter 4 being chimeric rabbit/human), plus the clinically establishedCD20-targeting monoclonal antibody products MabThera (rituximab; Roche,H01241308), Arzerra (ofatumumab; Novartis; C656294) and Gazyva(obinutuzumab, GA101; Roche, D287-41A GACD20) was tested in vitro usingBurkitt's lymphoma Raji cells. Raji cells (ATCC, Cat No. CCL-86) werecultured in RPMI 1640 supplemented with 10% heat-inactivated FBS, 1 U/mLpenicillin, 1 μg/mL streptomycin, and 4 mM L-glutamine. 0.1×10⁶ Rajicells were pre-incubated with antibodies in a total volume of 80 μLRPMI/0.2% BSA per well for 15 min on a shaker at RT. Next, NHS was addedto the pre-incubated cells to a final volume of 100 μL (final antibodyconcentrations 10 μg/mL; 20% NHS) and incubated for 45 minutes at 37° C.For all tested total antibody concentrations, different ratios of thetwo antibodies in the mixtures were tested (1:0-3:1-1:1-1:3-0:1). Plateswere centrifuged and cells were resuspended in 30 μL PI (2 μg/mL).Killing was calculated as the fraction PI-positive cells (%) determinedby flow cytometry on an iQue screener (Intellicyt). Data were analyzedand plotted using GraphPad Prism software.

The mixtures of the tested CD37 antibodies with an Fc-Fc interactionenhancing mutation and clinically established CD20 antibody productsshowed enhanced dose-dependent CDC activity compared to the sameconcentration of the single antibodies on Raji cells (FIG. 8). There waslittle difference in CDC activity at the different tested ratios of thetwo antibodies in the mixtures (1:3, 1:1 or 3:1). These data indicatethat the mixture of a hexamerization-enhanced CD37 antibody with anFc-Fc interaction enhancing mutation plus a clinically established CD20antibody product, such as MabThera, Arzerra (type I CD20 antibodies) orGazyva (type II CD20 antibody), may improve the therapeutic potentialfor patients with B cell malignancies, which frequently becomerefractory to standard CD20 targeted therapies alone.

The invention claimed is:
 1. A bispecific antibody comprising a first binding arm and a second binding arm, wherein the first binding arm comprises a first heavy chain and a first light chain comprising a first antigen-binding region, and the second binding arm comprises a second heavy chain and a second light chain comprising a second antigen-binding region, wherein both the first antigen-binding region and second antigen-binding region bind to human CD37, and wherein (a) the first antigen-binding region comprises a variable heavy chain (VH) region comprising the complementarity determining regions (CDRs) and the framework regions (FRs) within the amino acid sequence set forth in SEQ ID NO: 22 and a variable light chain (VL) region comprising the CDRs and FRs within the amino acid sequence set forth in SEQ ID NO: 29, and (b) the second antigen-binding region comprises a VH region comprising the CDRs and FRs within the amino acid sequence set forth in SEQ ID NO: 15 and a VL region comprising the CDRs and FRs within the amino acid sequence set forth in SEQ ID NO: 19, and wherein in each of the VH and VL regions, the CDRs and FRs are arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
 2. The bispecific antibody of claim 1, wherein the first binding arm comprises a first human IgG1 heavy chain constant region and the second binding arm comprises a second human IgG1 heavy chain constant region.
 3. The bispecific antibody of claim 1, wherein, in the first heavy chain, the amino acid residues corresponding to E430 and F405 in a human IgG1 heavy chain according to EU numbering are glycine and leucine, respectively; wherein, in the second heavy chain, the amino acid residues corresponding to E430 and K409 in a human IgG1 heavy chain according to EU numbering are glycine and arginine, respectively; and wherein the first light chain and second light chain comprise a light chain constant region consisting of the amino acid sequence set forth in SEQ ID NO:
 61. 4. The bispecific antibody of claim 1, wherein, in the first heavy chain, the amino acid residues corresponding to E430 and K409 in a human IgG1 heavy chain according to EU numbering are glycine and arginine, respectively; wherein, in the second heavy chain, the amino acid residues corresponding to E430 and F405 in a human IgG1 heavy chain according to EU numbering are glycine and leucine, respectively; and wherein the first light chain and second light chain comprise a light chain constant region consisting of the amino acid sequence set forth in SEQ ID NO:
 61. 5. The bispecific antibody of claim 1, wherein the first heavy chain comprises a first human IgG1 heavy chain constant region consisting of the amino acid sequence set forth in SEQ ID NO: 59, and the second heavy chain comprises a second IgG1 heavy chain constant region consisting of the amino acid sequence set forth in SEQ ID NO:
 60. 6. The bispecific antibody of claim 1, wherein the first heavy chain comprises a first human IgG1 heavy chain constant region consisting of the amino acid sequence set forth in SEQ ID NO: 60, and the second heavy chain comprises a second IgG1 heavy chain constant region consisting of the amino acid sequence set forth in SEQ ID NO:
 59. 7. The bispecific antibody of claim 1, wherein both the first binding arm and second binding arm comprise a human kappa light chain constant region.
 8. The bispecific antibody of claim 7, wherein the light chain constant region consists of the amino acid sequence set forth in SEQ ID NO:
 61. 9. The bispecific antibody of claim 1, which is a full-length antibody.
 10. The bispecific antibody of claim 1, wherein the first heavy and light chains and the second heavy and light chains are all inter-connected by disulfide bonds.
 11. A bispecific antibody comprising a first binding arm and a second binding arm, wherein the first binding arm comprises a first heavy chain and a first light chain comprising a first antigen-binding region, and the second binding arm comprises a second heavy chain and a second light chain comprising a second antigen-binding region, wherein both the first antigen-binding region and second antigen-binding region bind to human CD37, and wherein (a) the first antigen-binding region comprises a variable heavy chain (VH) region consisting of the amino acid sequence set forth in SEQ ID NO: 22 and a variable light chain (VL) region consisting of the amino acid sequence set forth in SEQ ID NO: 29, and (b) the second antigen-binding region comprises a VH region consisting of the amino acid sequence set forth in SEQ ID NO: 15 and a VL region consisting of the amino acid sequence set forth in SEQ ID NO:
 19. 12. The bispecific antibody of claim 11, wherein the first heavy chain comprises a first human IgG1 heavy chain constant region and the second heavy chain comprises a second human IgG1 heavy chain constant region.
 13. The bispecific antibody of claim 11, wherein both the first binding arm and second binding arm comprise a human kappa light chain constant region.
 14. The bispecific antibody of claim 11, wherein, in the first heavy chain, the amino acid residues corresponding to E430 and F405 in a human IgG1 heavy chain according to EU numbering are glycine and leucine, respectively; wherein, in the second heavy chain, the amino acid residues corresponding to E430 and K409 in a human IgG1 heavy chain according to EU numbering are glycine and arginine, respectively; and wherein the first light chain and second light chain comprise a light chain constant region consisting of the amino acid sequence set forth in SEQ ID NO:
 61. 15. The bispecific antibody of claim 11, wherein, in the first heavy chain, the amino acid residues corresponding to E430 and K409 in a human IgG1 heavy chain according to EU numbering are glycine and arginine, respectively; wherein, in the second heavy chain, the amino acid residues corresponding to E430 and F405 in a human IgG1 heavy chain according to EU numbering are glycine and leucine, respectively; and wherein the first light chain and second light chain comprise a light chain constant region consisting of the amino acid sequence set forth in SEQ ID NO:
 61. 16. The bispecific antibody of claim 11, wherein the first heavy chain comprises a first human IgG1 heavy chain constant region consisting of the amino acid sequence set forth in SEQ ID NO: 59, and the second heavy chain comprises a second IgG1 heavy chain constant region consisting of the amino acid sequence set forth in SEQ ID NO:
 60. 17. The bispecific antibody of claim 11, wherein the first heavy chain comprises a first human IgG1 heavy chain constant region consisting of the amino acid sequence set forth in SEQ ID NO: 60, and the second heavy chain comprises a second IgG1 heavy chain constant region consisting of the amino acid sequence set forth in SEQ ID NO:
 59. 18. The bispecific antibody of claim 11, which is a full-length antibody.
 19. The bispecific antibody according to claim 11, wherein the first heavy and light chains and the second heavy and light chains are all inter-connected by disulfide bonds.
 20. A bispecific antibody comprising a first binding arm and a second binding arm, wherein the first binding arm comprises a first heavy chain and a first light chain comprising a first antigen-binding region, and the second binding arm comprises a second heavy chain and a second light chain comprising a second antigen-binding region, wherein both the first antigen-binding region and second antigen-binding region bind to human CD37, and wherein (a) the first antigen-binding region comprises a variable heavy chain (VH) region consisting of the amino acid sequence set forth in SEQ ID NO: 22 and a variable light chain (VL) region consisting of the amino acid sequence set forth in SEQ ID NO: 29, and (b) the second antigen-binding region comprises a VH region consisting of the amino acid sequence set forth in SEQ ID NO: 15 and a VL region consisting of the amino acid sequence set forth in SEQ ID NO: 19, wherein the bispecific antibody comprises a first human IgG1 heavy chain constant region consisting of the amino acid sequence set forth in SEQ ID NO: 59 and a second human IgG1 heavy chain constant region consisting of the amino acid sequence set forth in SEQ ID NO: 60, and wherein the first light chain and second light chain comprise a light chain constant region consisting of the amino acid sequence set forth in SEQ ID NO:
 61. 21. The bispecific antibody of claim 20, wherein the first heavy and light chains and the second heavy and light chains are all inter-connected by disulfide bonds.
 22. A bispecific antibody comprising a first binding arm and a second binding arm, wherein the first binding arm comprises a first heavy chain and a first light chain comprising a first antigen-binding region, and the second binding arm comprises a second heavy chain and a second light chain comprising a second antigen-binding region, wherein both the first antigen-binding region and second antigen-binding region bind to human CD37, and wherein (a) the first antigen-binding region comprises a variable heavy chain (VH) region consisting of the amino acid sequence set forth in SEQ ID NO: 22 and a variable light chain (VL) region consisting of the amino acid sequence set forth in SEQ ID NO: 29, and (b) the second antigen-binding region comprises a VH region consisting of the amino acid sequence set forth in SEQ ID NO: 15 and a VL region consisting of the amino acid sequence set forth in SEQ ID NO: 19, wherein the bispecific antibody comprises a first human IgG1 heavy chain constant region consisting of the amino acid sequence set forth in SEQ ID NO: 60 and a second human IgG1 heavy chain constant region consisting of the amino acid sequence set forth in SEQ ID NO: 59, and wherein the first light chain and second light chain comprise a light chain constant region consisting of the amino acid sequence set forth in SEQ ID NO:
 61. 23. The bispecific antibody of claim 22, wherein the first heavy and light chains and the second heavy and light chains are all inter-connected by disulfide bonds. 